The influence of analogs of xanthine on the mutagenic effect of ultraviolet light

Summary Eight compounds can be added to those purine analogs which give synergistic effects with ultraviolet light: 1,3,7,9-tetra-methylxanthine methyl sulfate, 8-chlorocaffeine, ethyltheophylline, 6-dimethylaminopurine, 6-methoxypurine, 8-ethoxycaffeine, 1,3-dimethyl uric acid and 3-methyl uric acid. It appears likely that these are merely representatives of the class of methyl purines, and that other compounds are also active. The synergistic activity varies among the compounds investigated; some give a synergistic effect slightly higher than caffeine, but apparently act by the same mechanism. The height of effect of all of the synergistic purine analogs has been correlated with the stability of the structures determined by inductive effects and steric hindrance. The lack of activity with pyrimidines (l-methyl thymine and 1,3-dimethyluracil) leads to the conclusion that both rings (7,8,9 positions) of the purine molecule are necessary for the synergistic effect.This work was supported by the National Institutes of Health Research Grant AI-05340, the University of Kansas General Research Fund, and an N.I.H. Training Grant (5 T1 GM-703) to the Department of Microbiology.     

Enhancement and reduction by methylated oxypurines of the frequencies of chromatid aberrations induced by camptothecin in root-tip cells of Vicia faba.

In root-tip cells of Vicia faba the frequencies of chromatid aberrations induced by 3-h treatments with 0.05 microM camptothecin were strongly modified when the treatments were carried out in the presence of caffeine at concentrations above 1 mM. Depending on the concentration of caffeine, the clastogenic effect of camptothecin was either enhanced or reduced. At concentrations between 1 and 6 mM, caffeine increased the camptothecin-induced chromosome damage, the strongest enhancement being obtained at 5 mM. A reduction of the chromosome damage was apparent at caffeine concentrations above 10 mM, and in the presence of 20 mM caffeine the clastogenic effect of camptothecin was almost completely suppressed. When present during the camptothecin treatment, theophylline, 8-chlorocaffeine and 1,3,7,9-tetramethyluric acid influenced the induced chromosome damage in a similar way as caffeine, although with varying efficiency. If the concentrations required to produce the two types of modifying effect are used as a criterion, 8-chlorocaffeine was the most effective and 1,3,7,9-tetramethyluric acid the least, whereas caffeine and theophylline were about equally effective.     

Studies on DNA binding of caffeine and derivatives: evidence of intercalation by DNA-unwinding experiments

Caffeine and derivatives are compounds with pleiotropic effects on the genetic material which are supposed to originate from drugs binding to DNA. Here we show, by using two different topological methods, that methylated oxypurines, at biologically relevant concentrations, unwind DNA in a fashion similar to known intercalators. Methylated oxypurines could be ranked by decreasing unwinding potency: 8-methoxycaffeine greater than 8-ethoxycaffeine greater than 8-chlorocaffeine greater than caffeine greater than theophylline. These findings confirm, with a different assay, interaction of caffeine with DNA and add additional support to an intercalative mode of binding of these drugs to DNA.     

Effect of caffeine on repair of UV-premutations in phr- mutants of E. coli

Summary The frequency of uv-induced S3-mutations (resistance to 3 Streptomycin/ml) in E. coli B/phr^–/MC₂ was not significantly increased by postincubation in NB with caffeine, though an increase is to be expected if caffeine would inhibit the dark repair of the S3-premutations. The frequency was even decreased by high (0,1%) caffeine concentrations (Fig. 1), which indicates an enhancement of the (caffeine-resistant) repair. This enhancement may be caused indirectly by the observed prolongation by caffeine of the lag phase which gives more time for repair. Also the strong photoprotection and (indirect) photoreversion of the S3-mutations in this (non-photoreactivable) strain were not influenced by caffeine-posttreatment (Fig. 2 and 3). Thus, the dark repair assumed to be stimulated by pre- or post-illumination would be of the caffeine-resistant type. The repair of S3-premutations occurring during post-treatment in saline was inhibited by caffeine (Fig. 4). Also the dark-reactivation of cells killed by uv was inhibited by caffeine in the NB-agarmedium (Fig. 5). It is assumed that the repair of S3-premutations going on in NB-suspended cells is due to a mechanism which is not or only weakly inhibitable by caffeine and which is different from the caffeine-sensitive mechanism working under hunger conditions (perhaps by excising uv-products). Since reactivation of killed cells is caffeine-sensitive but reversion of S3-premutations is caffeine-resistant in NB-cells the uv-induced lethal lesions must be different from the S3-premutations.     

UV survival of human mycoplasmas: evidence of dark reactivation in Mycoplasma buccale.

The inactivation by ultraviolet (UV) light irradiation of mycoplasma cells of five human strains was monitored by investigating the colony-forming ability. The survival curves of five strains tested indicated that the cells of Mycoplasma buccale only are single and homogenously susceptible to UV light. The effect of the repair inhibitor, caffeine, on the colony-forming ability of UV-irradiated cells was investigated with M. buccale because of its homogenous susceptibility to UV light. The colony formation of irradiated cells was markedly depressed by post-irradiation treatment with caffeine at concentrations that had little or no effect on the colony formation of unirradiated cells. The colony-forming units (CFU) of UV-irradiated cells which were kept in broth without caffeine in the dark increased without a lag as the time in the dark increased. The colony-forming ability of the irradiated cells completely recovered after 3 hr in the dark. However, when irradiated cells were kept in the presence of caffeine, no increase in their CFU was observed. The mode of action of caffeine on UV-irradiated cells closely resembles that described for other organisms which possess dark reactivation systems for UV-induced damage in deoxyribonucleic acid (DNA). Thus, the results obtained provide evidence for the existence of a dark repair function in M. buccale.     

Survival-related DNA repair phenomena in cultured rat-kangaroo cells

Cultured cells (line PtK-2) from the marsupial mammal rat-kangaroo, or potoroo (Potorous tridactylis), which photoreactivate (PR) both UV-induced dimers and lethality, excise few dimers, and are only slightly sensitized by post-UV exposure to caffeine, were subjected to caffeine and hydroxyurea (HU) treatments during the 30-min PR period. It was found that neither caffeine nor HU inhibited PR of lethality as measured by colony-forming ability. Further, the cells exhibited no photoprotective properties and 3 mM caffeine potentiated the same slight survival decrease in both photoreactivated and unphotoreactivated cells. It is evident that caffeine does not inhibit PR or the survival-related dark repair systems to any great extent, and hence the caffeine-sensitive post-UV dark repair found in this (and possibly other) mammalian cell lines may not be related directly to survival-dependent pyrimidine dimer removal, but instead to lesions or repair processes other than, but not excluding, pyrimidine dimers.     

Enhancement of ultraviolet-induced mutation in bacteria by caffeine

Summary The addition of caffeine or theophylline to the growth medium of irradiatedE. coli B/rtry ^– resulted in a 10-fold or greater increase in the frequency oftry ⁺ mutants. These observations extend those ofWitkin (1958). Caffeine produced a slight reduction in the rate of RNA and protein synthesis, and a somewhat greater but temporary reduction in the rate of DNA synthesis. The analogue must be added immediately after UV-irradiation to produce its optimal effect, and the ability of an irradiated culture to respond to caffeine was lost completely after 20 min incubation in broth. Normal purine ribosides did not compete with caffeine. The optimal exposure time to caffeine was correlated with the time of DNA doubling, but marked increases of mutation frequency resulted when caffeine was present for 30 min in the absence of DNA synthesis. Incubation in caffeine before irradiation had no effect. Caffeine also reduced mutation frequency decline caused by incubation of irradiated bacteria in chloramphenicol. It is suggested that caffeine interfers with a dark repair enzyme system which removes a UV photoproduct (s) whose presence during DNA synthesis leads to mutation.With 4 Figures in the TextDedicated to ProfessorL. C. Dunn.Research supported by Grant NSF-G 14 044 from the National Science Foundation.     

Ultraviolet Irradiation of the Vegetative Cells of Dictyostelium discoideum

Experiments on the effect of ultraviolet (UV) light on the survival of vegetative Dictyostelium discoideum cells indicate that this is a relatively UV-resistant organism. Several factors suggest the presence of some type of repair process. Experiments to test for liquid-holding recovery and simple photoreactivation yielded negative results. Acriflavine and caffeine were utilized to possibly interfere with dark repair. Acriflavine produced no UV sensitization, but caffeine did cause a concentration-dependent decrease in survival of irradiated cells. When UV-irradiated cells were illuminated with photoreactivating light while suspended in caffeine, the survival increased above that for cells treated with caffeine alone, suggesting an overlap between lesions repaired by photorepair and dark repair. Growth experiments showed that UV light induced a dose-dependent division delay, followed by a period of retarded growth characterized by the presence of a constant fraction of nonviable cells in the irradiated population. The delayed exposure of cells to caffeine after irradiation showed that the magnitude of the caffeine sensitization diminished throughout the division-delay period. An action spectrum indicated probable nucleoprotein involvement in the induction of division delay. UV light retarded ribonucleic acid and protein synthesis and temporarily blocked deoxyribonucleic acid synthesis. However, synthesis of all three accelerated prior to the end of the division-delay period and then closely paralleled the increase in cell number.     

Repair of UV-induced damage in wild-type and mutant strains of Schizophyllum commune

The basidiomycete fungus Schizophyllum commune was found to have both photo-repair and dark-repair systems for UV-induced damage. Three UV-sensitive mutants were isolated and characterized for ability to repair UV-induced damage in light and dark, and for cross-sensitivity to caffeine and methyl methanesulfonate. Two of the mutants were damaged, to different extents, in their capacity for excision repair; one of these mutants was also probably damaged in post-replication repair. The third mutant was damaged only in post-replication repair.     

Characterization of Postreplication Repair in Mutagen-Sensitive Strains of DROSOPHILA MELANOGASTER

Mutants of Drosophila melanogaster, with suspected repair deficiencies, were analyzed for their capacity to repair damage induced by X-rays and UV radiation. Analysis was performed on cell cultures derived from embryos of homozygous mutant stocks. Postreplication repair following UV radiation has been analyzed in mutant stocks derived from a total of ten complementation groups. Cultures were irradiated, pulse-labeled, and incubated in the dark prior to analysis by alkaline sucrose gradient centrifugation. Kinetics of the molecular weight increase in newly synthesized DNA were assayed after cells had been incubated in the presence or absence of caffeine. Two separate pathways of postreplication repair have been tentatively identified by mutants derived from four complementation groups. The proposed caffeine sensitive pathway (CAS) is defined by mutants which also disrupt meiosis. The second pathway (CIS) is caffeine insensitive and is not yet associated with meiotic functions. All mutants deficient in postreplication repair are also sensitive to nitrogen mustard. The mutants investigated display a normal capacity to repair single-strand breaks induced in DNA by X-rays, although two may possess a reduced capacity to repair damage caused by localized incorporation of high specific activity thymidine-³H. The data have been employed to construct a model for repair of UV-induced damage in Drosophila DNA. Implications of the model for DNA repair in mammals are discussed.     

Quantitative and structural characteristics of lipids in Chlorobium and Chloroflexus

Exposure of dark grown resting Euglena to light induced the synthesis of chloroplast valyl-tRNA synthetase. Ethanol, a specific inhibitor of Euglena chloroplast development had little effect on chloroplast valyl-tRNA synthetase induction during the first 12 h of light exposure. Ethanol, however, completely inhibited enzyme synthesis between 12–72 h of light exposure. Malate, an alternative carbon source, had little effect on the photoinduction of valyl-tRNA synthetase. When dark grown resting cells were exposed to 2 h of light and returned to the dark, chloroplast valyl-tRNA synthetase continued to accumulate for 8–12 h at a rate which was less than the rate in cells maintained continuously in the light. The mutant strain W₃BUL lacks detectable chloroplast DNA and phototransformable protochlorophyllide, but retains a plastid remnant. Exposure of strain W₃BUL to light induced the synthesis of chloroplast valyl-tRNA synthetase and enzyme induction was not inhibited by ethanol. After 72 h of light exposure in the presence or absence of ethanol, enzyme levels in strain W₃BUL were comparable to the levels found in the wildtype strain after 8–14 h of light exposure. These results suggest that the nonchloroplast photoreceptor regulates the initial phase of enzyme synthesis. Mutant strain W₁₀BSmL differs from strain W₃BUL in that the plastid remnant if present, is greatly reduced. Chloroplast valyl-tRNA synthetase was undetectable in the strain W₁₀BSmL suggesting that the levels of active, cytoplasmically synthesized, chloroplast localized enzymes may be related to the developmental status of the chloroplast through the extent to which the enzyme precursor can be accumulated and or posttranslationally processed into an active enzyme within the chloroplast or chloroplast remnant.This research was supported by National Institutes of Health Grant GM26994, Biomedical support grant RR-0755 and funds from the Research Council, University of Nebraska     

The effect of inhibitors of DNA repair on the genetic instability of Streptomyces cattleya

Various streptomycetes show well defined instabilities that do not appear to be attributable to plasmid loss. The unstable phenotype, in many cases, arises at frequencies too high to be explained by point mutations. The frequency of instability can be enhanced by UV irradiation. Two major repair systems have been found in Escherichia coli: the 'error-free' system which is inhibited by caffeine and the 'error-prone' system which is inhibited by arsenite. Using spores of Streptomyces cattleya NRRL 8057 and the virulent actinophage VC11 we have shown that a caffeine inhibitable, host mediated UV repair system is active in spores during early development. Some evidence was also found for the presence of an arsenite inhibitable UV repair system. The caffeine inhibitable UV repair system was found to be involved in the induction of genetic instability in S. cattleya. The arsenite system may be implicated in the repair of such events. Genetic instability was also induced by single strand breaks in DNA caused by 32P.     

The effect of caffeine on repair in Chlamydomonas reinhardtii: I. Enhancement of recombination repair

The effect of caffeine on repair was studied in the green alga Chlamydomonas reinhardtii. Treatment of UV-irradiated wild-type (UVS⁺) cells with a sublethal level of caffeine caused a significant increase in survival compared to untreated UV-irradiated cells. Caffeine did not affect survival in the repair-deficient strain UVSE1, which is deficient in repair of UV-induced damage carried out by enzymes associated with recombination during meiosis. A significant increase in survival in the presence of caffeine was observed in the repair-deficient strain UVSE4 in which recombination during meiosis is not affected. Treatment of zygotes homozygous for UVS⁺, UVSE1, or UVSE4 with sublethal levels of caffeine caused marked increases in recombination frequency in UVS⁺ and UVSE4 zygotes and no increase in recombination in UVSE1 zygotes. These results indicate that caffeine increases recombination in normal strains. Increased opportunity for recombination caused by caffeine would not result in increased recombination frequency in the UVSE1 strain, assuming limited-recombination enzyme activity in this strain. The observed increase in survival following UV-irradiation in the presence of caffeine in strains having normal recombination would therefore be associated with a caffeine-induced increase in opportunities for recombination repair.     

Electron microscope study on the meiotic cycle of Acanthopachylus aculeatus (Arachnida; Opiliones)

Summary The primary spermatocytes of an Opilionid, Acanthopachylus aculeatus show (at least as a rule) two bodies which in longitudinal sections appear as integrated of up to 12 dark bands each about 300 Å wide, interlaced with one another by a curtain of thin filaments. The same bodies appear in transversal sections as formed by an hexagonal lattice the nodal points of which are the cross sections of the dark bands. The study of spermatocytes at early prophase demonstrated that at the inception these bodies are formed by association of several structures comparable to those observed in the axis of paired autosomes and called synaptinemal complexes.The findings in this species are compared with their similar in other species, particularly with species of Gryllidae.This investigation was supported by United States Public Health Service Research Grant GM 08337 from the Research Grants Branch, Division of Medical Sciences, and partly by Grant RF 61034 from The Rockefeller Foundation.     

Induction of alkaline phosphatase activity in HeLa cells by sodium butyrate

Summary The induction of HeLa cell alkaline phosphatase activity by sodium butyrate could be inhibited by the coadministration of caffeine or theophylline. The inhibitions were dose dependent, and at any given concentration the potency was theophylline > caffeine. Although the induction by sodium butyrate was more sensitive to the inhibition by the xanthines than was that produced by 5-iodo-2′-deoxyuridine, the magnitudes of the increases in cyclic AMP concentrations after treatment with the xanthines were similar in the inhibition of both types of induction. The induction of alkaline phosphatase activity by sodium butyrate also produced a shift in the thermostability pattern of the enzyme, with a proportionately greater increase in the heat-labile, rather than heat-stable, from of the activity. Supported by National Cancer Institute Grant CA16460.     

Caffeine- and amino acid-effects upon try(plus) revertant yield in U.V.-irradiated hcr(plus) and hcr (minus) mutants of E. coli B-r

Summary The influence of supplementation of the post-irradiation plating medium with caffeine and/or amino acids, upon both U.V.-induced killing and try⁺ reversion yield, has been studied in hcr⁺ and hcr^- derivatives of E. coli B/r tryptophan auxotroph WWP-2. All experiments have been carried out with stationary phase cells grown in aerated nutrient broth.In the hcr^- strain, caffeine causes no enhancement of either killing or try⁺ revertant yield. There is mutational enhancement by supplementation with a low level of tryptophan, and an even greater effect when supplementation is with tryptophan plus an additional pool of other amino acids.In the hcr⁺ strain, tryptophan and/or a pool of other amino acids cause an enhancement of try⁺ yield, as in the hcr^- strain. Caffeine causes lethal enhancement on several different media. There is an apparently straightforward dose enhancement by caffeine, of lethality and try⁺ revertant frequency, on media containing no, or only, tryptophan. On media containing, however, both a low level of tryptophan and an additional pool of other amino acids, caffeine causes a preferential enhancement of try⁺ revertant frequency over and above pure dose enhancement.These results suggest that U.V. may cause two types of lesion. One type may lead only to mutation, and is repaired by both the caffeine-insensitive MFD, and the caffeine-sensitive hcr, dark repair systems. This first type of lesion is protected from repair by the presence of a pool of amino acids. The second type of lesion is hypothesised to be capable of causing either lethality or mutation, is repaired only by the hcr dark-repair system, and is not subject to protection by an amino acid pool.This work was initiated at the Radiological Research Laboratories, Columbia University, New York.     

Lesch-Nyhan syndrome: Absence of the mutant enzyme in erythrocytes of a heterozygote for both normal and mutant hypoxanthine-guanine phosphoribosyl transferase

Subjects heterozygous for the Lesch-Nyhan syndrome with a deficiency of the X-linked gene for the enzyme hypoxanthine-guanine phosphoribosyl transferase (PRT) would be expected to have two populations of erythrocytes in roughly equal proportions—one type with the normal enzyme and the other type exhibiting the mutant form of the enzyme. In contrast to this prediction, previous studies utilizing an X-linked gene for another enzyme as a marker for the PRT locus have suggested that erythrocytes from heterozygotes consist largely of cells with the normal form of the enzyme. We have recently described a mutant form of hypoxanthine-guanine phosphoribosyl tranferase with altered kinetic properties which allow it to be measured in artificial mixtures with the normal enzyme. The mutant enzyme could not be detected in erythrocyte lysates from a proven heterozygote for both the normal and this mutant form of the enzyme. This provides additional evidence that either inactivation of the X-chromosome in erythropoietic tissue from the heterozygote for PRT deficiency is not random or that random X-chromosome inactivation is followed by selection against erythrocyte precursors with the mutant enzyme.This study was supported in part by USPHS Research Grant No. AM14362, USPHS Training Grant No. AM05620, and a grant (RR-30) from the General Clinical Research Centers Program of the Division of Research Resources, National Institutes of Health.     

The light/dark cycle operation with an hour-scale period enhances caffeine production by Coffea arabica cells

The intermittent light irradiation with an hour-scale period is used for producing caffeine by Coffea arabica cells. Three factors concerning the light/dark cycle operation such as light intensity, the length of the cycle (period), and the ratio of the illumination time to the dark time (light/dark ratio) were investigated to optimize the caffeine production efficiency regarding light consumption. The light/dark ratio of 1/1 enhanced caffeine production, reaching the same level as continuous light; thus, the intermittent light irradiation improved the production efficiency twofold. The production was not influenced by the period, but was determined by light intensity regardless of intermittent or continuous light irradiation.     

Purification and characterization of a novel caffeine oxidase from Alcaligenes species

Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO(3), 0.4%; KH(2)PO(4), 0.15%; Na(2)HPO(4), 0.05%; FeCl(3).6H(2)O, 0.0005%; CaCl(2).2H(2)O, 0.001%; MgSO(4).7H(2)O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 degrees C had maximal activity at pH 7.5 and 35 degrees C. The purified caffeine oxidase had strict substrate specificity towards caffeine (K(m) 8.94 microM and V(max) 47.62 U mg protein(-1)) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca(+2), Mg(+2) and Na(+), but was completely inhibited by Co(+2), Cu(+2) and Mn(+2) at 1mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.     

Acid phosphatase in the Golgi apparatus of cells forming extracellular matrix of hard tissues

Summary Histochemical studies using cryostat sections of fixed rodent fetal and newborn tissues indicated that acid phosphatase (APase) staining of the Golgi apparatus (GA) of cells secreting matrix for hard tissue formation was a general phenomenon. The enzyme was chiefly observed in the GA of tall secretory ameloblasts involved in enamel formation and in the GA of odontoblasts forming dentine; lysosome-like granules reactive for this enzyme were also observed in these cells. Activity was also intense in the GA and lysosomes of osteoblasts involved in intramembranous and endochondral bone formation.High levels of APase in the GA of extracellular matrix-forming cells appeared to correlate with secretory activity. The GA of most other cells, even chondroblasts forming cartilage matrix, had much less marked APase activity. Contrary to previous suggestions, it appears that APase may have a more direct role in osteogenesis than the osteolytic or resorptive action usually cited.This investigation was supported by PHS Research Grant No. DE 02668 from the National Institute of Dental Research and in part by General Research Support Grant No. RR 5333 from the General Research Support Branch of the National Institutes of Health.The authors gratefully acknowledge the excellent technical assistance of Dorothy H. Clapp and Peggy E. Yates.