Mutagenic and epigenetic influence of caffeine on the frequencies of UV-induced ouabain-resistant Chinese hamster cells

Caffeine, given as a post-treatment to UV-irradiated Chinese hamster cells in vitro, modified the frequency of induced mutations at the ouabain resistance locus. Mutation frequencies were increased when caffeine was added only for the DNA repair and mutation fixation period. When caffeine was added after the DNA repair and mutation fixation period, or immediately after DNA damage and for the entire repair and selection period, mutation frequencies were reduced. A hypothesis, given to explain both results, is that caffeine, by blocking a constitutive “error-free” postreplication repair process, allows an “error-prone” DNA repair process to produce many mutations. Moreover, caffeine, possibly by modifying C-AMP metabolism, causes a repression of induced mutations which, in effect, explains its anti-mutagenic and anti-carcinogenic properties.     

Chromosome and DNA damage and their repair in higher plants irradiated with short-wave ultraviolet light

In this review the authors present only their own results. They include the determination of the duration of the different stages of the cell cylce in UV-irradiated barley cells, the effect of different UV doses on the frequency of chromosome aberrations in barley, the increase in UV-induced chromosome aberration frequency induced in barley by caffeine and the effect of UV doses on the induction of pyrimidine dimers and sites sensitive to UV-endonuclease action (ESS) in barley cells and Nicotina tabacum protoplasts. In addition, the excision of pyrimidine dimers and ESS after irradiation with various doses of UV, unscheduled DNA synthesis in N. tabacum protoplasts and the correlation between the induction of pyrimidine dimers in DNA and the frequency of chromosome aberrations are reported. Data demonstrating that photoreactivation decrease the number of DNA lesions and chromosome aberrations induced by UV are also presented.     

Caffeine delays replication fork progression and enhances UV-induced homologous recombination in Chinese hamster cell lines

The ability to bypass DNA lesions encountered during replication is important in order to maintain cell viability and avoid genomic instability. Exposure of mammalian cells to UV-irradiation induces the formation of DNA lesions that stall replication forks. In order to restore replication, different bypass mechanisms are operating, previously named post-replication repair. Translesion DNA synthesis is performed by low-fidelity polymerases, which can replicate across damaged sites. The nature of lesions and of polymerases involved influences the resulting frequency of mutations. Homologous recombination represents an alternative pathway for the rescue of stalled replication forks. Caffeine has long been recognized to influence post-replication repair, although the mechanism is not identified. Here, we found that caffeine delays the progress of replication forks in UV-irradiated Chinese hamster cells. The length of this enhanced delay was similar in wild-type cells and in cell deficient in either homologous recombination or nucleotide excision repair. Furthermore, caffeine attenuated the frequency of UV-induced mutations in the hprt gene, whereas the frequency of recombination, monitored in this same gene, was enhanced. These observations indicate that in cells exposed to UV-light, caffeine inhibits the rescue of stalled replication forks by translesion DNA synthesis, thereby causing a switch to bypass via homologous recombination. The biological consequence of the former pathway is mutations, while the latter results in chromosomal aberrations.     

Retarded excision of pyrimidine dimers in human unstimulated lymphocytes

Using immuno-labelling of cyclobutane pyrimidine dimers (CPDs) in nuclei of peripheral lymphocytes after their UVC-irradiation and cultivation, we have found that within the first four hours of cultivation the CPD-specific fluorescent signal from cell nuclei increased. Earlier, a similar increase in binding of antibody specific for pyrimidine (6-4) pyrimidone photoproducts to undenatured DNA isolated from UV-irradiated Chinese hamster ovary cells was reported (Mitchell et al., 1986). Our experiments showed that nucleotide excision repair enzyme might induce such of DNA modification in lymphocyte nuclei that increased specific antibody binding to DNA fragments with lesions. We suggest that enzymatic formation of open structures in DNA predominated qualitatively over dual-incision and excision of these fragments, and resulted in the enhanced exposure of the pyrimidine dimers in nuclei to specific antibodies. The results evidence that nucleotid excision repair in unstimualted human lymphocytes being deficient in dual incision and removal of UV-induced DNA lesions appear to be capable of performing chromatin relaxation and pre-incision uncoiling of DNA fragments with lesions.     

Mechanism of Caffeine Enhancement of Mutations Induced by Sublethal Ultraviolet Dosages

Certain chemical compounds increase mutation frequency of Escherichia coli B/r significantly when used in conjunction with nonlethal ultraviolet (UV) dosages. Studies were done to elucidate the mechanism of this enhancing mutational effect. Dark survival curves showed that 500 μg of caffeine per ml in the postirradiation medium markedly decreased survival to 60 ergs/mm² of UV in strain B/r. Caffeine did not markedly decrease survival to UV in strain B/r WP-2 hcr⁻. At least 90% of the mutations induced to streptomycin resistance by UV and 85% of those induced by UV with caffeine could be photoreversed. Experiments with thymine analogues suggested that thymine dimerization at the streptomycin locus was the primary premutational photoproduct induced by sublethal UV dosages. Caffeine did not interfere with the photoreversal of induced mutants, indicating that it probably does not bind to the photoreactivating enzyme or to a UV-induced lesion in the DNA. Addition of DNA or irradiated DNA with 500 μg of caffeine per ml resulted in no loss of the caffeine activity. The excision of UV-induced thymine-containing dimers from E. coli B/r T⁻ was investigated in the presence and absence of caffeine. Our results indicated that caffeine prevents excision of thymine dimers, presumably by binding to the excising enzyme. This binding results in an impairment of repair, which produces the increase in mutant numbers.     

Aberrant DNA repair and enhanced mutagenesis following mutagen treatment of Chinese hamster Ade−C cells in a state of purine deprivation

Ade⁻C is a Chinese hamster ovary cell line auxotrophic for purines because of a mutation in the de novo synthetic pathway. We now show that, in the absence of exogenous hypoxanthine, replicative DNA synthesis is rapidly shut down. Various aspects of DNA repair have been studied in purine-starved cells. Incision, the first step of excision repair of UV damage, appears normal, as do the later steps, repair synthesis (demonstrated following chemical damage as well as UV-irradiation) and ligation. However, removal of UV-induced pyrimidine dimers is not detected, and it seems that the repair that occurs is aberrant. This behaviour is associated with an increase in cell killing by UV light, and a several-fold increase in the frequency of mutations induced by UV.     

Inhibition of post-replication repair of alkylated DNA by caffeine in Chinese hamster cells but not HeLa cells

Caffeine has been found to potentiate the lethal effects of sulphur mustard (SM) and N-methyl-N-nitrosourea (MNU) in a line of Chinese hamster cells but not in a line of HeLa cells. The sensitization of SM-treated cells by caffeine was S phase specific, and persisted for up to 24 h after alkylation of asynchronous cell cultures. The sensitization of MNU-treated cells, however, was not S phase specific but persisted for up to 50 h after the initial alkylation. Possible explanations for this difference between these two types of alkylating agent were discussed. Previously, evidence was presented which suggested that the alkylation-induced delay in the time of the peak rate of DNA synthesis in Chinese hamster cells was associated with the operation of post-DNA replication repair mechanism in these cells. Caffeine has now been found to reverse this alkylation-induced delay of DNA synthesis in both SM- and MNU-alkylated Chinese hamster cells. It is therefore proposed that caffeine sensitizes alkylated cells by inhibition of a post-replication DNA repair mechanism. No support was obtained for the alternative possibility that caffeine inhibits alkylation-induced excision repair of damaged DNA. The role of DNA repair in the production of the lethal mutagenic and cytological effects of alkylating agents is discussed.     

The effect of methylated oxypurines on the size of newly-synthesized DNA and on the production of chromosome aberrations after UV irradiation in Chinese hamster cells.

A comparison has been made, in Chinese hamster cells, of the ability of various methylated oxypurines to inhibit post-replication repair of DNA after UV irradiation and to potentiate UV-induced chromosome aberrations. DNA synthesized in UV-irradiated cells contains gaps, which are subsequently sealed by a process termed post-replication repair. In rodent cells this process is inhibited by caffeine and its analogues. This has been quantitated by measuring the molecular weight of the DNA synthesized in UV-irradiated cells during a 4-h pulse-labelling period in the presence or absence of inhibitors--the lower molecular weight the greater the inhibition. Eight methylated oxypurines were tested; caffeine and chlorocaffeine were always the most potent inhibitors, tetramethyluric acid was inactive, and the other five derivatives (methoxycaffeine, ethoxycaffeine, paraxanthine, theobromine and theophylline) had intermediate effects. Measurements of the potentiation of UV-induced chromosome aberrations showed that treatments with caffeine or chlorocaffeine again had the greatest effects, tetramethyluric acid and also theophylline had no potentiating activity, and methoxycaffeine was intermediate. This correlation between effects at the molecular and cytological levels is consistent with the hypothesis that the inhibition of post-replication repair by methylated oxypurines gives rise to the increased production of chromosome aberrations.     

Expression of a mammalian DNA photolyase confers light-dependent repair activity and reduces mutations of UV-irradiated shuttle vectors in xeroderma pigmentosum cells

Photoreactivation is one of the DNA repair mechanisms to remove UV lesions from cellular DNA with a function of the DNA photolyase and visible light. Two types of photolyase specific for cyclobutane pyrimidine dimers (CPD) and for pyrimidine (6-4) pyrimidones (6-4PD) are found in nature, but neither is present in cells from placental mammals. To investigate the effect of the CPD-specific photolyase on killing and mutations induced by UV, we expressed a marsupial DNA photolyase in DNA repair-deficient group A xeroderma pigmentosum (XP-A) cells. Expression of the photolyase and visible light irradiation removed CPD from cellular DNA and elevated survival of the UV-irradiated XP-A cells, and also reduced mutation frequencies of UV-irradiated shuttle vector plasmids replicating in XP-A cells. The survival of UV-irradiated cells and mutation frequencies of UV-irradiated plasmids were not completely restored to the unirradiated levels by the removal of CPD. These results suggest that both CPD and other UV damage, probably 6-4PD, can lead to cell killing and mutations.     

Persistence of pyrimidine dimers during post-replication repair in ultraviolet light-irradiated Escherichia coli K12

We have used a new assay for pyrimidine dimers to obtain evidence regarding the mechanism of post-replication repair of ultraviolet light-induced damage in excision-deficient (uvr) mutants of Escherichia coli. Our data indicate that dimers are gradually removed from the irradiated DNA under conditions permitting post-replication repair. Concomitantly, dimers appear in daughter strands synthesized after irradiation. The daughter strands initially contain gaps. During post-replication repair the gaps are filled and the originally discontinuous DNA is joined into long molecules resembling those observed in unirradiated control cells. Density transfer experiments reported by other investigators have provided evidence that the gap-filling involves exchanges between irradiated parental DNA and unirradiated daughter strands. The results of our experiments are in accord with this possibility and suggest that some dimers are included in the exchanged regions. Our data imply that intact, dimer-free DNA molecules are not necessarily generated by gap-filling and may not appear in uvr cells until several hours after u.v. irradiation. Instead, dimers may be gradually diluted among successive generations of DNA molecules synthesized after irradiation.     

Determination of Pyrimidine Dimers in Escherichia coli and Cryptosporidium parvum during UV Light Inactivation, Photoreactivation, and Dark Repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

Daughter DNA synthesis in UV-resistant and UV-sensitive Chinese hamster clones cells under UV irradiation

By means of ultracentrifugation in alkaline sucrose gradients it has been shown that the size of DNA fragments synthesized in Chinese hamster cells of UV-sensitive clone (CHS-1) after exposure to UV light was equal to the distance between pyrimidine dimers in the parental DNA determined using endonuclease of Micrococcus luteus. With the UV-resistant clone (V-79), the length of fragments of the newly synthesized DNA was much longer than that between pyrimidine dimers in the parental DNA. The data obtained support the model according which DNA synthesis on the UV-irradiated template gives rise to gaps opposite to pyrimidine dimers.     

Effect of Caffeine on Postreplication Repair in Human Cells

DNA synthesized shortly after ultraviolet (UV) irradiation of human cells is made in segments that are smaller than normal, but at long times after irradiation the segments made are normal in size. Upon incubation, both the shorter and the normal segments are elongated and joined by the insertion of exogenous nucleotides to form high molecular weight DNA as in nonirradiated cells. These processes occur in normal human cells, where UV-induced pyrimidine dimers are excised, as well as in xeroderma pigmentosum (XP) cells, where dimers are not excised. The effect of caffeine on these processes was determined for both normal human and XP cells. Caffeine, which binds to denatured regions of DNA, inhibited DNA chain elongation and joining in irradiated XP cells but not in irradiated normal human or nonirradiated cells. Caffeine also caused an alteration in the ability to recover synthesis of DNA of normal size at long times after irradiation in XP cells but not in normal cells.     

(6-4) photoproducts and not cyclobutane pyrimidine dimers are the main UV-induced mutagenic lesions in Chinese hamster cells.

A partial revertant (RH1-26) of the UV-sensitive Chinese hamster V79 cell mutant V-H1 (complementation group 2) was isolated and characterized. It was used to analyze the mutagenic potency of the 2 major UV-induced lesions, cyclobutane pyrimidine dimers and (6-4) photoproducts. Both V-H1 and RH1-26 did not repair pyrimidine dimers measured in the genome overall as well as in the active hprt gene. Repair of (6-4) photoproducts from the genome overall was slower in V-H1 than in wild-type V79 cells, but was restored to normal in RH1-26. Although V-H1 cells have a 7-fold enhanced mutagenicity, RH1-26 cells, despite the absence of pyrimidine dimer repair, have a slightly lower level of UV-induced mutagenesis than observed in wild-type V79 cells. The molecular nature of hprt mutations and the DNA-strand specificity were similar in V79 and RH1-26 cells but different from that of V-H1 cells. Since in RH1-26 as well as in V79 cells most hprt mutations were induced by lesions in the non-transcribed DNA strand, in contrast to the transcribed DNA strand in V-H1, the observed mutation-strand bias suggests that normally (6-4) photoproducts are preferentially repaired in the transcribed DNA strand. The dramatic influence of the impaired (6-4) photoproduct repair in V-H1 on UV-induced mutability and the molecular nature of hprt mutations indicate that the (6-4) photoproduct is the main UV-induced mutagenic lesion.     

UV-induced immobilization of DNA repair protein XPA in different human cell line

XPA repair protein is absolutely needed for nucleotide excision repair (NER). It preferentially binds UV-irradiated DNA in vitro and possibly takes place in the recognition of pyrimidine dimers, the main type of UV-lesions in DNA. Using immunofluorescent microscopy and immunoblotting technique we have found that XPA protein is fully extractable by Triton X-100 solution from non-irradiated normal human fibroblasts, but after UV-irradiation its extractability decreases in UV-dose dependent manner. UV-induced XPA-immobilization was observed in human cell lines with different types of repair defects, but XPA-extractability from unirradiated cells of these lines was significantly lower in comparison with normal fibroblasts. These data do not permit to make conclusion concerning the distinct connection of this phenomenon with different pathways of NER. Histone deacetylase inhibitor, sodium butyrate, did not change the level of extractability in unirradiated and UV-irradiated normal human cells and CHO cells, defective in global genome repair, that indicated the independence of XPA-immobilization from the level of histone acetylation. It was established with the help of confocal microscopy that XPA-foci in detergent-treated UV-irradiated cell were partially colocalized with the focal sites of PCNA, an auxiliary protein of DNA polymerases delta and epsilon. It may mean that a part of detergent-resistant XPA foci correspond to DNA repair synthesis sites, but the major part of immobilized XPA reflects the early step of repair proteins assembly formation needed for the repair of the lesions.     

UV-induced DNA degradation in Aspergillus nidulans cells]

UV-induced DNA degradation was studied in mycellial cells of Aspergillus nidulans wild type and several uvs mutants. It was shown to be an enzymatic specific process which possibly reflects the excision of pyrimidine dimers from UV-damaged DNA. Inhibition of DNA degradation by caffeine and 2,4-dinitrophenol shows the connection between degradation and repair of DNA. Two ways of DNA degradation were found in A. nidulans cells, one of them being glucose dependent and the other--glucose independent. The dependence of DNA degradation on protein synthesis before and after UV-irradiation was demonstrated. The scheme of ways of DNA degradation and its genetic control were suggested on the basis of uvs mutations effect on UV-induced DNA degradation.     

Photoreactivation and repair replication in rat kangaroo cells

Potorous tridactylis cells can perform photoreactivation, i.e., the visible light- catalyzed reversal of UV-induced pyrimidine dimers in DNA. UV-induced inhibitions of total RNA and DNA synthesis can also be partially reversed by exposure to visible light. P. tridactylis cells can also perform repair replication, but the extent of the latter is reduced if the cells are exposed to visible light (VL). None of these effects are observed in mouse L cells, which cannot perform photoreactivation. The results are consistent with the concept of pyrimidine dimers are one of the main substrates for repair replication.     

A study of the effect of caffeine upon excision repair of damaged DNA.

Cultured mammalian cells incur damage to their DNA when exposed to ultraviolet light or adduct-producing mutagens such as 4-nitroquinoline-1-oxide (4NQO). At least two processes are important in repair of such damage: post-replication repair and excision repair. Many researchers have reported that caffeine inhibits the former process, which occurs in connection with semiconservative DNA replication, especially in rodent cell lines such as mouse lymphoma or Chinese hamster. Excision repair is not generally considered caffeine-sensitive, although the data are somewhat conflicting because some studies had used rodent cells, which show little or no excision repair, or human cells in which alternate repair processes may have been operating.Human peripherhal blood lymphocytes from healthy donors were treated with UV light or 4NQO in order to produce pyrimidine dimers or adducts. Caffeine at concentrations of 0.75–3.0 mM was included in some cultures. The cells treated with caffeine were incubated for 90 min prior to mutagen treatment and for the entire period thereafter until cell harvests. [³H]Thymidine was added and the uptake quantitated as a measure of DNA repair. DNA replication was inhibited by hydroxyurea, so that only excision repair was measured by this method. Separate plates of cells not exposed to mutagens exhibited negligible or low thymidine uptakes.Following harvest, the cells were lysed and the DNA extracted. The DNA released was measured spectrophotometrically and then placed into liquid-scintillation counter (LSC) vials for measurement of incorporated radioactivity. Resulting cpm/μ DNA were compared for cells with and without caffeine. Lymphocytes from patients with systemic lupus erythematosus (SLE), who previously had demonstrated reduced levels of excision repair under these conditions, were also tested with caffeine. Caffeine did not inhibit repair by normal lymphocytes and the reduced repair seen in the SLE patients was not further reduced in its presence.In a series of pulse-chase experiments, some cells were treated with 4NQO and allowed to incubate with [³H]thymidine for 3 h and were harvested at the end of this period, while others were given a 13-h chase i n cold thymidine before harvest. The cpm/μg DNA for both groups were virtually identical, both in the presence and absence of 2.0 mM caffeine.     

General characteristics, molecular and genetic analysis of two new UV-sensitive mutants of Chlamydomonas reinhardtii

Two new UV-sensitive mutants of Chlamydomonas, UVS10 and UVS11, were isolated. Both behave as single nuclear mutations. UVS10 was mapped to linkage group I. UVS11 is a separate, unlinked mutation but has not yet been located to a specific linkage group. Both mutants are proficient in the excision of pyrimidine dimers from nuclear DNA. The survival of UV-irradiated UVS11 is increased when plated in the presence of 1.5 mM caffeine, similar to wild-type. Caffeine has no effect on the survival of UV-irradiated UVS10. UV-irradiated UVS11 frequently divides at least once before dying, in contrast to UVS10 or wild-type. UVS11 also exhibits a much increased frequency of mutation to streptomycin resistance after UV irradiation.     

Induction and repair of DNA strand breaks in Bacteroides fragilis

Alkaline sucrose gradient sedimentation was used to establish whether strand breakage and repair take place in the DNA of UV-irradiated Bacteroides fragilis during the removal of pyrimidine dimers. A B. fragilis wild-type strain and two of its repair mutants, a mitomycin C sensitive mutant (MTC25) having wild-type levels of UV survival, and a UV-sensitive, mitomycin C sensitive mutant (UVS9), were investigated. Under anaerobic conditions, far-UV irradiation induced metabolically regulated strand breakage and resynthesis in the wild-type strain, but this was markedly reduced in both the MTC25 and UVS9 mutants. Approximately half of the strand breaks generated by the various strains were rejoined during further holding in buffer. Under replicating conditions, complete repair of strand breaks in the wild type was observed. Caffeine treatment under anaerobic conditions caused direct DNA strand breakage in B. fragilis cells but did not inhibit UV-induced breakage or repair.