Genetic effects of specific DNA lesions in mammalian cells

Accurate dosimetry for chemical mutagens is extremely difficult, and precise manipulation of the frequency of a particular lesion is ordinarily impossible. With 8-MOP plus UVA, however, both are possible because 8-MOP, when photoactivated by one photon of UVA, forms monoadducts whilst crosslinks are formed only if a second photon of light photoactivates the monoadducts. If 8-MOP molecules that are unreacted after a UVA exposure are removed from cells by washing, the effect of a subsequent UVA irradiation can be attributed only to the conversion of monoadducts to DNA interstrand crosslinks. Using this experimental procedure and L5178Y mouse lymphoma cells, we have shown that DNA interstrand crosslinks are at least 10-fold more effective at causing both sister-chromatid exchanges and chromosomal aberrations than are monoadducts. In contrast, crosslinks are no more effective than monoadducts in mutation induction. These experiments identify directly for the first time that a particular chemically induced lesion, DNA interstrand crosslinks, can, like thymine dimers, cause chromosomal aberrations and sister-chromatid exchanges. The results also show that sister-chromatid exchanges can be induced independently of mutations.     

Mutation induction and killing of Escherichia coli by DNA adducts and crosslinks: a photobiological study with 8-methoxypsoralen.

Low doses of 350 nm radiation (NUV) in the presence of 8-methoxypsoralen (8-MOP) induce predominantly mono-adducts in bacterial DNA. Further exposure to NUV in the absence of 8-MOP converts a proportion of these mono-adducts to interstrand cross-links. Using this approach the relative effects of adducts and cross-links on bacteria with different repair capacities was studied. Escherichia coli WP100 uvrA recA, believed to be totally deficient in the ability to repair 8-MOP plus NUV damage to DNA, was inactivated on average by a single photon event occurring with a quantum efficiency of about 0.03. We conclude that the inactivating lesion is probably a single mono-adduct. E. coli WP2 uvrA, deficient in excision endonuclease activity, may be inactivated by a very small number of cross-links, probably one. These conclusions are consistent with present knowledge of the repair capabilities of these bacteria. Conversion of mono-adducts to cross-links in WP2 uvrA (which occurs with a quantum efficiency of around 0.3) greatly increases lethality but results in a reduction of the induced mutation frequency presumably because cross-links are (almost) invariably lethal. In the repair-proficient strain WP2 both adducts and cross-links can be repaired but the latter are more likely than the former to lead to either death or mutation.     

Mitomycin C-induced damage and repair in human and pig lymphocytes

Human and pig lymphocytes were used to compare the chromosomal sensitivity to MMC and the efficiency of repair of MMC-induced DNA adducts. No significant interspecies differences were found. The results obtained show that SCE frequencies are linearly correlated with MMC doses. During the G0 period there are indications that lymphocytes may half-repair the DNA-interstrand crosslinks transforming bi- into mono-adducts. SCEs induced by MMC decrease to near control levels in the second cell cycle. Therefore, most MMC lesions responsible for SCEs should be repaired between the moment in the first S phase in which they induce the exchanges and the onset of the second S period.     

Fate of photo-induced 8-methoxypsoralen mono-adducts in yeast. Evidence for bypass of these lesions in the absence of excision repair

A fraction of UVA-induced 8-methoxypsoralen (8-MOP) mono-adducts can be transformed by a second UVA (365 nm) irradiation procedure into lethal cross-links in Saccharomyces cerevisiae. To follow the fate of cross-linkable mono-adducts, cells were incubated in complete medium between the two UVA doses and survival was measured. The killing effect of the second UVA dose decreases rapidly in haploid wild-type as well as in strains blocked in mutagenic (RAD6+ type) or in recombinogenic (RAD52+ type) repair pathways. This is also true in the pso1-1 and pso2-1 strains selected for sensitivity to 8-MOP plus UVA treatment. In contrast, persistence of mono-adducts is observed in strains blocked in the excision-resynthesis repair pathway. In other words, cross-linkable mono-adducts are repaired by the excision process. The use of the cell-cycle conditional mutant strain (cdc14-1) permitted us to apply the second dose at a specific cell-cycle stage (post-G2 phase) after a 'priming' UVA treatment on stationary (G1) phase cells. Such experiments showed a bypass of mono-adducts in an excision-deficient context for at least one round of DNA replication.     

Repair of 8-methoxypsoralen monoadducts in mouse lymphoma cells

Studies of the repair of DNA lesions at biologically important doses is extremely difficult for most mutagens. With 8-methoxypsoralen (8-MOP) plus longwave ultraviolet light (UVA) as the lesion-inducing agent, however, it is easy to manipulate the relative frequency of different DNA adducts by means of a special experimental protocol (the tap-and-test protocol) and this can be used to measure repair of DNA adducts. Three classes of photoadducts are produced by 8-MOP plus UVA treatment: 3,4-cyclobutane monoadducts, 4',5'-cyclobutane monoadducts, and 8-MOP-DNA interstrand crosslinks. A monoadduct is formed when a photoactivated 8-MOP molecule reacts with a pyrimidine base. An 8-MOP-DNA interstrand crosslink is formed when an existing monoadduct is photoactivated to react with another pyrimidine base on the opposite DNA strand. Thus monoadducts are formed by absorption of one photon of light and crosslinks by absorption of two. In the tap-and-test experiments, cells were exposed to UVA in the presence of 8-MOP and then re-exposed to UVA in the absence of free 8-MOP so that only crosslinks can be produced by the second UVA treatment. By means of this technique we have previously shown that DNA crosslinks are much more effective than monoadducts at producing chromosomal damage (sister-chromatid exchanges and micronuclei) but not mutations (Liu-Lee et al., 1984). If L5178Y mouse lymphoma cells were able to remove monoadducts, incubation prior to the second UVA treatment should lead to decreases in the effect of re-irradiation, because fewer monoadducts would be available for crosslink formation. In this way, we have found that psoralen monoadducts are repaired in these cells and that about 70% of those capable of crosslink formation are removed or otherwise made unavailable for crosslink formation in 6 h.     

Can a mixed damage interfere with DNA‐prottein cross‐links repair?

Some photochemical and photobiological properties of 4,5',8-trimethylpsoralen (TMP) have been studied in comparison with 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2 one (FQ) and 8-methoxypsoralen (8-MOP). TMP and FQ can photobind to mammalian cell DNA in vivo , by UVA irradiation, forming DNA-protein cross-links (DPC), but only TMP shows a strong capacity of inducing interstrand cross-links (ISC). The mechanism of DPC formation was studied using the double irradiation method in Chinese hamster ovary (CHO) cells, and DPC were detected by alkaline elution. Both TMP and FQ induce covalent diadducts linking together DNA and proteins. Studying the formation of double strand breaks (DSB) in CHO cells we observed that TMP induced a low amount of DSB, similar to 8-MOP. TMP and 8-MOP induced chromosomal aberrations in CHO cells to the same extent, while FQ appeared to be more active. Our data suggest that the ISC induced by TMP could trap enzymes involved in DPC repair.     

In vitro crosslinking of DNA by 8-methoxypsoralen visualized by electron microscopy

By electron microscopic visualisation of totally denatured DNA, we have detected photochemically induced 8-methoxypsoralen crosslinks in vitro after irradiation at 360 nm. The amount of crosslinks was expressed as the percentage of DNA length which was kept in double-stranded appearance by closely situated crosslinks. This percentage correlated well with irradiation time, irradiation intensity, and the concentration of 8-methoxypsoralen. These parameters have also been correlated with the mean size and the size distribution of non-crosslinked regions of DNA, so called bubbles. For a comparison with another psoralen type, we have carried out a similar set of experiments using 4,5,8-trimethylpsoralen.     

Dictamnine,an alkaloid which crosslinks DNA in the presence of ultraviolet light

In the presence of ultraviolet light, the furoquinoline alkaloid, dictamnine, caused calf thymus DNA to become easily renaturable. The effect was less pronounced than for the furocoumarin, 8-methoxypsoralen. Ease of renaturation is evidence of the formation of interstrand crosslinks in DNA. The mechanism of crosslink formation by this alkaloid may be like that of 8-methoxypsoralen.     

Lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

The genotoxic effect of 8-methoxypsoralen damages (monoadducts and crosslinks) on plasmid DNA was studied. pBR322 DNA was treated with several concentrations of 8-methoxypsoralen plus fixed UVA light irradiation. After transformation into E. coli cells with different repair capacities (uvrA, recA and wild-type), plasmid survival and mutagenesis in ampicillin- and tetracycline-resistant genes were analysed. Results showed that crosslinks were extremely lethal in all 3 strains; indeed, it seemed that they were not repaired even in proficient bacteria. Monoadducts were also found to be lethal although they were removed to some extent by the excision-repair pathway (uvrA-dependent). Damaged plasmid DNA appeared to induce mutagenic repair, but only in the wild-type strain. In order to study the influence of the SOS response on plasmid recovery, preirradiation of the host cells was also performed. Preirradiation of the uvrA or wild-type strains significantly increased plasmid recovery. Consistent with the expectations of SOS repair, no effect was observed in preirradiated recA cells. Plasmid recovery in the excision-deficient strain was mainly achieved by the mutagenic repair of some fraction of the lesions, probably monoadducts. The greatest increase in plasmid recovery was found in the wild-type strain. This likely involved the repair of monoadducts and some fraction of the crosslinks. We conclude that repair in preirradiated repair-proficient cells is carried out mainly by an error-free pathway, suggesting enhancement of the excision repair promoted by the induction of SOS functions.     

DNA damage induced by carcinogenic lead chromate particles in cultured mammalian cells.

Particulate lead chromate is a highly water-insoluble cytotoxic and carcinogenic agent, but its mechanism of action remains obscure. We investigated its effects on DNA damage in CHO cells after a 24-h exposure using alkaline or neutral filter elution and cytogenetic studies. Concentrations (0.08, 0.4 and 0.8 micrograms/cm2), which reduced the colony-forming efficiency of CHO cells to 94, 50 and 10%, respectively, produced dose-dependent DNA single-strand breaks and DNA-protein crosslinks, but no DNA double-strand breaks or DNA-DNA crosslinks were observed. The single-strand breaks were absent from cells given a 24-h recovery period after removal of the treatment medium, even though most of the particles remained adhered to cells and to the culture dish. In contrast, both the DNA-protein crosslinks and chromosomal aberrations persisted even after the 24-h recovery period. These results suggest that the mechanism of the particle-induced early DNA single-strand breaks may be different from DNA-protein crosslinks and the lesions leading to chromosomal aberrations, or alternatively, that the repair of single-strand breaks is more efficient than the repair of DNA-protein crosslinks in the unavoidable continuing presence of carcinogen. These results also suggest that the chromosome damage may be related to the persistent DNA-protein crosslinks, and further confirm the genotoxic activity of carcinogenic lead chromate particles.     

In vitro assessment of the toxicity of metal compounds

A review has been compiled illustrating the directions taken in examining the genotoxic effects of metals and their compounds centering only on those studies pertaining to effects of metals and their compounds on DNA structure and function, such as the induction of DNA strand breaks, production of DNA-protein crosslinks, induction of chromosomal aberrations, and sister chromatid exchanges. Although it is premature to declare a cause and effect relationship between the carcinogenic activity of metals and their ability to induce one or more lesions in DNA, strong evidence is emerging to suggest such a relationship. Low concentrations of metals induce the appearance of DNA lesions, such as strand breaks and crosslinks, or induce sister chromatid exchanges or DNA repair synthesis. Assays based upon these events constitute extremely sensitive probes for genotoxic effects of metals and their compounds. These effects of metals on DNA are consistent with the currently accepted mechanism of chemical carcinogenesis, allowing the acquisition and propagation of altered DNA function. The lack of complete information on the activity of metals in producing DNA lesions allow only preliminary conclusions to be drawn. Certain compounds containing potentially or actually carcinogenic elements, such as Ni, Be, As, Cr, Cd, and to a minor extent Pb, have yielded positive responses in one or more DNA lesion assays. At relatively nontoxic levels of Ni and Cr, considerable evidence suggests that multiple types of DNA lesions are induced.     

DNA crosslinks and DNA replication in mouse FM3A cells after treatment with 8-methoxypsoralen plus near-ultraviolet radiation

The rate of DNA-chain elongation was studied in mouse FM3A cells after treatment with 8-methoxypsoralen plus near-ultraviolet radiation using the minimal doses (1 μg/ml 8-methoxypsoralen plus 1–2.5 kJ/m² of near-ultraviolet radiation) which inhibited cell-cycle progression or DNA replication. A rapid decrease in incorporation of [³H]thymidine and recovery to some extent during incubation after treatment have been reported (Hyodo, M., Fujita, H., Suzuki, K., Yoshino, K., Matsuo, I. and Ohkido, M. (1982) Mutat. Res. 94, 199–211). The results of the present study showed that the rate was not changed suggesting that the decrease in [³H]thymidine incorporation was not due to the rate of DNA-chain elongation, but was due to change in the frequency of initiation of replication. Formation of DNA crosslinks was then studied by the sedimentation of pre-labeled DNA in an alkaline sucrose gradient. The results showed that, at these doses of 8-methoxypsoralen plus near-ultraviolet radiation, approx. 2–7 crosslinks were formed per 10⁹ Da. It was also suggested that some of the DNA crosslinks might be repaired during the prolonged incubation, but unrepaired crosslinks were still present after 24 h incubation.     

Differential sensitivity of Fanconi anaemia lymphocytes to the clastogenic action of cis-diamminedichloroplatinum (II) and trans-diamminedichloroplatinum (II)

Summary Fanconi anaemia (FA) lymphocytes were tested for their susceptibility to chromosomal breakage by cis-diamminedichloroplatinum (II) [cis-Pt(II)] and its stereoisomer trans-diamminedichloroplatinum (II) [trans-Pt(II)]. Unlike trans-Pt(II), which is a rather inefficient clastogen, cis-Pt(II) is very efficient in inducing chromosomal breakage in FA cells at concentrations that hardly affect control cells. As both cis-Pt(II) and trans-Pt(II) are capable of inducing DNA interstrand crosslinks but only cis-Pt(II) can induce DNA intra-strand crosslinks, this result suggests that FA cells may be specifically sensitive to the intrastrand type of DNA crosslink.     

Formation by bifunctional furocoumarins of DNA crosslinks and their repair in cultured mouse embryo fibroblasts

Formation of crosslinks in DNA by three bifunctional psoralen derivatives plus UVA light in mouse embryo fibroblasts was evaluated by a NaI density gradient centrifugation method. Psoralen was shown to be a more active cross-linking agent than 8-methoxypsoralen. As for 4,5',8-trimethylpsoralen, it needed much lower concentrations and much less 365 nm light fluence to yield high percentages of crosslinked DNA. Repair of adducts formed by these psoralen derivatives was studied by splitting the irradiation dose into two equal parts separated by variously long dark repair periods. It was shown that essentially only monoadducts formed during the first irradiation period were repaired. These mouse embryo fibroblasts seem unable to repair interstrand DNA crosslinks.     

DNA interstrand crosslink repair in mammalian cells

DNA damage by agents crosslinking the strands presents a formidable challenge to the cell to repair for survival and to repair accurately for maintenance of genetic information. It appears that repair of DNA crosslinks occurs in a path involving double strand breaks (DSBs) in the DNA. Mammalian cells have multiple systems involved in the repair response to such damage, including the Fanconi anemia pathway that appears to be directly involved, although the mechanisms and site of action remain elusive. A particular finding relating to deficiency of the Fanconi anemia pathway is the observation of chromosomal radial formations after ICL damage. The basis of formation of such chromosomal aberrations is unknown although they appear secondarily to DSBs. Here we review the processes involved in response to DNA interstrand crosslinks which might lead to radial formation and the role of the nucleotide excision repair gene, ERCC1, which is required for a normal response, not just to DNA crosslinks, but also for DSBs at collapsed replication forks caused by substrate depletion. J. Cell. Physiol. 220: 569–573, 2009. © 2009 Wiley‐Liss, Inc.     

Chromosome damage induced in human lymphocytes by 5-methoxypsoralen and 8-methoxypsoralen plus UV-A

The induction of structural chromosome aberrations and sister chromatid exchanges (SCE) was studied in human lymphocytes in vitro after treatment with the two bifunctional furocoumarins 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP) in the presence of UV-A. The results show that both psoralens induce a dose-dependent increase in the SCE rate as well as in structural chromosome aberrations. 5-MOP was 2.0-2.5 times more effective for the induction of chromosome breaks and had a slightly stronger effect with respect to SCE induction. A significant influence on proliferation kinetics could be observed only with 5-MOP plus UV-A.     

Maturation of nascent DNA fragment is disturbed after treatment of cultured mouse FM3A cells with 8-methoxypsoralen plus near-ultraviolet radiation

By the method of sedimentation in 5–20% alkaline sucrose gradient, the process of maturation of the nascent DNA fragment was studied with cultured mouse FM3A cells treated with 8-methoxypsoralen plus near-ultraviolet radiation. This treatment is known to cause crosslinks of the chromosomal DNA strands. The profile of the newly-replicated DNA, labeled for 10 min with [³H]thymidine immediately after treatment, was the same as that of the untreated cells, where the incorporated radioactivity was present in the intermediate DNA fragment (about 50–80 S). But, when the treated cells were labeled after several hours of incubation, the labeled DNA became much shorter due to inhibition of maturation of the initial DNA fragment (the Okazaki fragment) to the intermediate DNA. With the use of aphidicolin, a specific inhibitor of eukaryotic DNA polymerase α, it became apparent that, in addition to formation of the crosslinks, further DNA replication is required to cause this inhibition of DNA maturation. Aphidicolin also suppressed the inhibition of incorporation of [³H]thymidine into cellular DNA after treatment, but inhibition of this incorporation resumed after its removal.     

Structure of the Escherichia coli 16 S ribosomal RNA. Psoralen crosslinks and N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine crosslinks detected by electron microscopy

Escherichia coli 16 S ribosomal RNA in reconstitution buffer has been photochemically crosslinked with aminomethyltrimethylpsoralen and chemically crosslinked with N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine. The positions of crosslinking have been detected by viewing the molecules in the electron microscope. DNA restriction fragments that contain psoralen mono-adducts were hybridized and crosslinked to the samples so that the orientations of the crosslinked molecules were seen directly. A two-dimensional histogram method has been used to classify the different types of looped crosslinked molecules. These methods allow the identification of 13 distinct types of loops in the photochemically crosslinked molecules and 31 distinct types of loops in the chemically crosslinked molecules. The psoralen experiments are a reinvestigation of some of our earlier results. Some of the crosslinks were previously reported in the incorrect orientation; with the corrected orientation, seven of the psoralen crosslinks can now be correlated with complementarities in the proposed secondary-structure models. However, there are still six other psoralen crosslinks that indicate additional contacts not found in the current models. The chemical crosslinks indicate pairs of single-stranded regions that must be close in the folded molecule. Many of these crosslinks occur between regions that are distant in the secondary structure; these crosslinks indicate part of the three-dimensional form of the folded molecule.     

Mechanisms for chromosomal aberrations in mammalian cells

Experimental evidence is presented for the involvement of DNA double-strand breaks in the formation of radiation-induced chromosomal aberrations. When X-irradiated cells were post-treated with single-strand specific Neurospora crassa endonuclease (NE), the frequencies of all classes of aberration increased by about a factor 2. Under these conditions, the frequencies of DNA double-strand breaks induced by X-rays (as determined by neutral sucrose-gradient centrifugation), also increased by a factor of 2. The frequency of chromosomal aberrations induced by fast neutrons (which predominantly induce DNA double-strand breaks) was not influenced by post-treatment with NE. Inhibition of poly(ADP-ribose) polymerase, an enzyme that uses DNA with double-strand breaks as an optimal template, by 3-aminobenzamide also increased the frequencies of X-ray-induced chromosomal aberrations, which supports the idea that DNA double-strand breaks are important lesions for the production of chromosomal aberrations induced by ionizing radiation.