Alkaline gel electrophoresis of deoxyribonucleic acid photoreacted with trimethylpsoralen: rapid and sensitive detection of interstrand cross-links

Restriction fragments of phage lambda and phi X174 deoxyribonucleic acid (DNA) were photoreacted with 4,5',8-trimethylpsoralen to various extents, and the amount of covalent cross-linking was determined by electron microscopy of the DNA under totally denaturing conditions. The DNA was then analyzed by electrophoresis in alkaline agarose gels. A single cross-link in a DNA molecule produced a large decrease in its electrophoretic mobility. With DNA fragments 0.3--4 kilobase pairs in size, the apparent Mr (molecular weight) of the cross-linked DNA was 2.0 +/- 0.1 times and Mr of the unreacted, single-stranded DNA. A single cross-link in a larger DNA molecule resulted in an even greater increase in apparent Mr. Further cross-linking produced a decrease in the apparent Mr of the DNA, reaching a plateau at a value of 1.4 +/- 0.1 times the Mr of the unreacted, single-stranded DNA over a large range of fragment sizes (0.6--10 kilobase pairs). The apparent Mr of the cross-linked DNA was weakly dependent on the percentage of agarose in the gel. Although highly sensitive to interstrand cross-links the electrophoretic mobilities appeared to be unaffected by low levels of monoadducts (trimethylpsoralen covalently bound to one strand of the DNA). The DNA bandwidths increased by as much as 4-fold at low extents of cross-linking, presumably due to heterogeneity in the locations of the cross-links in the DNA molecules. The bands became sharp again at high levels of reaction. These observations from the basis of a new assay for interstrand DNA cross-links that is both more sensitive and more convenient than previous methods.     

Systemic suppression of contact hypersensitivity by ultraviolet b radiation or methoxsalen/ultraviolet a radiation in the guinea pig

Treatment of strain 2 guinea pigs with ultraviolet b (uvb) (280-320 nm) radiation or methoxsalen, followed by ultraviolet a (uva) (320-400 nm) radiation, decreased the contact hypersensitivity (CHS) reaction to sensitizing agents applied subsequently to unirradiated sites. The decreased reactivity could be transferred to syngeneic animals and appeared to be caused by antigen-specific suppressor T lymphocytes. Ultraviolet b irradiation of sensitized animals did not affect elicitation of CHS in unirradiated skin.     

N(4)C-ethyl-N(4)C cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of different orientations.

The preparation and physical properties of short DNA duplexes that contain a N(4)C-ethyl-N(4)C interstrand cross-link are described. Duplexes that contain an interstrand cross-link between mismatched C-C residues and duplexes in which the C residues of a -CG- or -GC- step are linked to give "staggered" interstrand cross-links were prepared using a novel N(4)C-ethyl-N(4)C phosphoramidite reagent. Duplexes with the C-C mismatch cross-link have UV thermal transition temperatures that are 25 degrees C higher than the melting temperatures of control duplexes in which the cross-link is replaced with a G-C base pair. It appears that this cross-link stabilizes adjacent base pairs and does not perturb the structure of the helix, a conclusion that is supported by the CD spectrum of this duplex and by molecular models. An even higher level of stabilization, 49 degrees C, is seen with the duplex that contains a -CG- staggered cross-link. Molecular models suggest that this cross-link may induce propeller twisting in the cross-linked base pairs, and the CD spectrum of this duplex exhibits an unusual negative band at 298 nm, although the remainder of the spectrum is similar to that of B-form DNA. Mismatched C-C or -CG- staggered cross-linked duplexes that have complementary overhanging ends can undergo self-ligation catalyzed by T4 DNA ligase. Analysis of the ligated oligomers by nondenaturing polyacrylamide gel electrophoresis shows that the resulting oligomers migrate in a manner similar to that of a mixture of non-cross-linked control oligomers and suggests that these cross-links do not result in significant bending of the helix. However, the orientation of the staggered cross-link can have a significant effect on the structure and stability of the cross-linked duplex. Thus, the thermal stability of the duplex that contains a -GC- staggered cross-link is 10 degrees C lower than the melting temperature of the control, non-cross-linked duplex. Unlike the -CG- staggered cross-link, in which the cross-linked base pairs can still maintain hydrogen bond contacts, molecular models suggest that formation of the -GC- staggered cross-link disrupts hydrogen bonding and may also perturb adjacent base pairs leading to an overall reduction in helix stability. Duplexes with specifically positioned and oriented cross-links can be used as substrates to study DNA repair mechanisms.     

Probing DNA interstrand cross-link formation by an oxidized abasic site using nonnative nucleotides

The C4'-oxidized abasic site (C4-AP) forms two types of interstrand cross-links with the adjacent nucleotides in DNA. Previous experiments revealed that dG does not react with the lesion and that formation of one type of cross-link is catalyzed by the opposing dA. iso-Guanosine·dC and 2-aminopurine·dT base pairs were used to determine why dG does not cross-link with C4-AP despite its well known reactivity with other bis-electrophiles. 7-Deaza-2'-deoxyadenosine was used to probe the role of the nucleotide opposite C4-AP in the catalysis of interstrand cross-link formation.     

Phototherapy and photopharmacology

The activation of 8-methoxypsoralen (8-MOP) by long-wavelength ultraviolet A light (UVA, 320-400 nm) induces the formation of interstrand cross-links in DNA. Psoralen plus UVA (PUVA) is widely used in the treatment of psoriasis, a hyperproliferative disease of the skin. A new psoralen plus UVA therapy has been developed in which the 8-MOP-containing blood of cutaneous T cell lymphoma (CTCL) patients is irradiated with UVA light extracorporeally (i.e., extracorporeal photopheresis). The first group of patients had the leukemic variant of CTCL. A regimen of two treatments on successive days at monthly intervals produced a clinical response in eight of 11 patients. In this review the properties of several psoralens (both naturally occurring and synthetic derivatives) are compared, using several assays (DNA cross-linking, inhibition of lymphocyte response to mitogen stimulation, and cell viability). The development of a panel of monoclonal antibodies that recognize 8-MOP-modified DNA is also described. These antibodies have been used to quantitate 8-MOP photoadduct levels in human DNA samples. In addition to the psoralens, the light activation of two other compounds, gilvocarcin and an insulin-psoralen conjugate, is described.     

Repair of cross-linked DNA and survival of Escherichia coli treated with psoralen and light: effects of mutations influencing genetic recombination and DNA metabolism.

Repair of cross-linked DNA was studied in Escherichia coli strains carrying mutations affecting DNA metabolism. In wild-type cells, DNA strands cut during cross-link removal were rejoined during a subsequent incubation into high-molecular-weight molecules. This rejoining was dependent on gene products involved in genetic recombination. A close correlation was found relating recombination proficiency, the rate of strand rejoining, and formation of viable progeny after DNA cross-linking by treatment with psoralen and light. Wild-type cells and other mutants which were Rec+ (sbcB, recL, recL sbcB, recB recC sbcA, recB recC sbcB, xthA1, and xthA11) rejoined cut DNA strands at a rate of 0.8 +/- 0.1 min -1 at 37 degrees C and survived 53 to 71 cross-links per chromosome. recB, recC, recB recC, recF, or polA strains showed reduced rates of strand rejoining and survived 4 to 13 cross-links per chromosome. Recombination-deficient strains (recA, recB recC sbcB recF, recB recL) and lexA failed to rejoin DNA strands after crosslink removal and were unable to form colonies after treatments producing as few as one to two cross-links per chromosome. Strand rejoining occurred normally in cells with mutations affecting DNA replication (dnaA, danB, dnaG, and dnaE) under both permissive and nonpermissive conditions for chromosome replication. In a polA polB dnaE strain strand rejoining occurred at 32 degree C but not at 42 degree C, indicating that some DNA synthesis was required for formation of intact recombinant molecules.     

Chromosome damage in Chinese hamster cells sensitized to near-ultraviolet light by psoralen and angelicin

The clastogenic effect of furocoumarins psoralen and angelicin in the presence of near-UV (320-380 nm) differs greatly, as do their modes of interaction with DNA. Psoralen, which requires only one-fifth as much light energy to produce the same lethal effect as angelicin at equimolar concentrations, is able to cross-link DNA whereas angelicin cannot. The frequency of micronuclei which arise from chromosomal fragments shows the same differential effect as lethality. Indeed aberrations account for much or all of the lethality observed. Metaphase analysis at comparable aberration frequencies revealed that angelicin and psoralen both induce chromatid deletions and a wide spectrum of chromatid exchanges. These data show that both cross-links and monoadducts to the DNA can result in chromosomal aberrations. The relative contributions of cross-links and monoadducts to chromosomal aberrations still remain to be determined. It is noteworthy that extensive chromosomal damage is induced in mammalian cells by the combination of psoralen and near-UV, a treatment which is currently widely used in the therapy of psoriasis.     

Direct photoaffinity labeling by dolastatin 10 of the amino-terminal peptide of beta-tubulin containing cysteine 12

Tubulin with bound [5-3H]dolastatin 10 was exposed to ultraviolet light, and 8-10% of the bound drug cross-linked to the protein, most of it specifically. The primary cross-link was to the peptide spanning amino acid residues 2-31 of beta-tubulin, but the specific amino acid could not be identified. Indirect studies indicated that cross-link formation occurred between cysteine 12 and the thiazole moiety of dolastatin 10. An equipotent analog of dolastatin 10, lacking the thiazole ring, did not form an ultraviolet light-induced cross-link to beta-tubulin. Preillumination of tubulin with ultraviolet light, known to induce cross-link formation between cysteine 12 and exchangeable site nucleotide, inhibited the binding of [5-3H]dolastatin 10 and cross-link formation more potently than it inhibited the binding of colchicine or vinblastine to tubulin. Conversely, binding of dolastatin 10 to tubulin inhibited formation of the cross-link between cysteine 12 and the exchangeable site nucleotide. Dithiothreitol inhibited formation of the beta-tubulin/dolastatin 10 cross-link but not the beta-tubulin/exchangeable site nucleotide cross-link. Modeling studies revealed a highly favored binding site for dolastatin 10 at the + end of beta-tubulin in proximity to the exchangeable site GDP. Computational docking of an energy-minimized dolastatin 10 conformation at this site placed the thiazole ring of dolastatin 10 8-9 A from the sulfur atom of cysteine 12. Dolastatin 15 and cryptophycin 1 could also be docked into positions that overlapped more extensively with the docked dolastatin 10 than with each other. This result was consistent with the observed binding properties of these peptides.     

Inactivation of Escherichia coli, F′ Episomes at Transfer, and Bacteriophage Lambda by Psoralen Plus 360-nm Light: Significance of Deoxyribonucleic Acid Cross-Links

We have investigated some biological consequences of light-induced psoralen-deoxyribonucleic acid (DNA) adducts and find that for several Escherichia coli functions (killing of strain AB2480 recA13 uvrA6, inactivation of phage lambda plaque-forming ability in wild type and uvrA6 hosts, loss of ability to transmit intact Flac(+) episomes), a light exposure sufficient for production of a single cross-link per DNA molecule correlates well with the biological consequence. Although one cross-link per genome is apparently lethal to recA13 uvr(-) strains, mutants carrying the recA13 or uvrA6 markers survive light exposures producing 6.7 and 16 cross-links per genome, respectively, and wild-type cells recover from 65 psoralen cross-links. Evidently, the excision and recombinational repair systems complement one another in reconstructing an intact genome from cellular DNA containing psoralen photoproducts. The above bacterial and phage strains, in which DNA repair processes are minimized, are also extremely sensitive to pyrimidine dimer-forming 254-nm UV light (without psoralen), and were expected to respond similarly to formation of psoralen-pyrimidine base monoadducts in their DNA. Since the biological inactivation by psoralen correlates well with cross-link formation, we suggest that the sensitizing action of this drug primarily derives from its ability to form DNA cross-links.     

Structure, flexibility, and repair of two different orientations of the same alkyl interstrand DNA cross-link

Interstrand DNA cross-links are the principal cytotoxic lesions produced by chemotherapeutic bifunctional alkylating agents. Using an N(4)C-ethyl-N(4)C interstrand DNA cross-link to mimic this class of clinically important cancer chemotherapeutic agents, we have characterized the repair, structure, and flexibility of DNA that contains this cross-link in two different orientations. Plasmid DNAs in which the cytosines of single CpG or GpC steps are covalently linked were efficiently processed by repair proficient and homologous recombination deficient strains of Escherichia coli. Repair in a nucleotide excision repair (NER) deficient strain was less efficient overall and displayed a 4-fold difference between the two cross-link orientations. Both the structure and flexibility of DNA containing these cross-links were examined using a combination of (1)H NMR, restrained molecular dynamics simulations, and atomic force microscopy (AFM). The NMR structure of a decamer containing a CpG interstrand cross-link shows the cross-link easily accommodated within the duplex with no disruption of hydrogen bonding and only minor perturbations of helical parameters. In contrast, disruptions caused by the GpC cross-link produced considerable conformational flexibility that precluded structure determination by NMR. AFM imaging of cross-link-containing plasmid DNA showed that the increased flexibility observed in the GpC cross-link persists when it is embedded into much larger DNA fragments. These differences may account for the different repair efficiencies seen in NER deficient cells.     

Induction and repair of psoralen cross-links in DNA of normal human and xeroderma pigmentosum fibroblasts

Skin fibroblasts from normal human subjects were exposed in vitro to long-wave ultraviolet radiation (UVA, 320–400 nm) alone, or in combination with 8-methoxypsoralen (8-MOP). DNA damage was analysed with the alkaline elution technique before and after post-treatment incubation of the cells at 37°C for various times.Cells treated with UVA at 1.1 J/cm² showed an increased DNA elution rate, which returned to the normal level within 30 min of post-treatment incubation. In cells treated with PUVA (8-MOP at 20 μg/ml plus UVA at 0.04 J/cm²), the alkaline elution rate was not different from untreated control cells, either before or after post-treatment incubation for times up to 7 days.When the PUVA treatment was followed first by a washing, to remove any unbound 8-MOP, and then by UVA (PUVA + UVA) at 1.1 J/cm², the alkaline elution rate decreased below the control level. During the post-treatment incubation of the PUVA + UVA-treated cells there was a gradual increase of the alkaline elution rate to a level significantly above that in control cells. This increase was observed after 30 min. It reached a miaximum after 24 h and remained after 7 days of post-treatment incubation. Cells from a patient with xeroderma pigmentosum of complementation group A, which were given the same PUVA + UVA treatment, did not show any change in the alkaline elution rate during the post-treatment incubation.If, as seems likely, an increased alkaline elution rate indicates an increase of DNA breaks, and a decreased alkaline elution rate indicates the sealing of breaks and/or the formation of cross-links, the results would suggest the following: (1) UVA irradiation in itself is capable of inducing DNA breaks, which are rapidly sealed during post-treatment incubation; (2) PUVA treatment induces mono-adducts, some of which appear to remain in the DNA for at least 7 days of post-treatment incubation and can be activated to form DNA cross-links by a second dose of UVA; (3) DNA cross-links induced by PUVA + UVA can be recognized by a repair process that involves the formation of DNA breaks. This process is not observed in xeroderma pigmentosum cells of group A.     

Growth and nitrogen utilization in seedlings of mountain birch (Betula pubescens ssp. tortuosa ) as affected by ultraviolet radiation (UV-A and UV-B) under laboratory and outdoor conditions

 Growth patterns and nitrogen economy were studied in pot-grown seedlings of mountain birch subjected to different ultraviolet radiation under both laboratory and outdoor conditions at Abisko in northern Sweden. In the laboratory, nutrient supply, temperature, humidity, ultraviolet radiation-A (UV-A, 320–400 nm) and B (UV-B, 280–320 nm) were controlled, while photosynthetically active radiation (PAR, 400–700 nm) and photoperiod varied naturally. Under outdoor conditions nutrient supply was controlled, and the irradiation treatments were ambient and above-ambient UV-B using additional fluorescent lamps. Mountain birch nitrogen economy was affected by increased ultraviolet radiation, as reflected by a changed relationship between plant growth and plant nitrogen both in the laboratory and outdoors. In the laboratory enhanced UV-A decreased leaf area per unit plant biomass (leaf area ratio) but increased biomass productivity, both per unit leaf area (leaf area productivity) and per unit leaf nitrogen (leaf nitrogen productivity). Low levels of UV-B affected growth patterns and nitrogen economy in a similar way to enhanced UV-A. High levels of UV-B clearly decreased relative growth rate and nitrogen productivity, as leaf area ratio, leaf area productivity and leaf nitrogen productivity were all decreased. Under outdoor conditions above-ambient levels of UV-B did not alter growth or biomass allocation traits of the seedlings, whilst nitrogen productivity was increased. Mountain birch seedlings originating from different mother trees varied significantly in their responses to different ultraviolet radiation. Received: 10 April 1997 / Accepted: 19 September 1997     

Base sequence-independent distorsions induced by interstrand cross-links in cis-diamminedichloroplatinum (II)-modified DNA.

Physico-chemical and immunological studies have been done in order to further characterize the distorsions induced in DNA by the interstrand cross-links formed between the antitumor drug cis-diamminedichloroplatinum (II) (cis-DDP) and two guanines on the opposite strands of DNA at the d(GC/GC) sites. Bending (45 degrees) and unwinding (79 +/- 4 degrees) were determined from the electrophoretic mobility of multimers of 21- 24-base pairs double-stranded oligonucleotides containing an interstrand cross-link in the central sequence d(TGCT/AGCA). The distorsions induced by the interstrand cross-link in the three 22-base pairs oligonucleotides d(TGCT/AGCA), d(AGCT/AGCT) and d(CGCT/AGCG) were compared by means of gel electrophoresis, circular dichroism, phenanthroline-copper footprinting and antibodies specifically directed against cis-DDP interstrand cross-links. The four different technical approaches indicate that the distorsions are independent of the chemical nature of the base pairs adjacent to the interstrand cross-link. The general conclusion is that the interstrand cross-link induces a bending and in particular an unwinding larger than other platinum adducts and the distorsions are independent of the nature of the bases (purine or pyrimidine) adjacent to the d(GC/GC) site.     

Production of active oxygen species (1O2 and O2-.) by psoralens and ultraviolet radiation (320-400 nm)

Furocoumarins (psoralens) are potent skin photosensitizing agents that are used in combination with long-wavelength ultraviolet radiation (320-400 nm) in the treatment of psoriasis and other skin diseases. Twelve linear and angular psoralens, capable of forming monofunctional and bifunctional adducts with DNA, were examined with a view to elucidate the role of 1O2 and O2-. in evoking skin photosensitization reactions and skin carcinogenesis. The results showed that both linear psoralens (capable of forming interstrand cross-links) and isopsoralens (angular, monofunctional type) and 3-carbethoxypsoralen (a linear and monofunctional type) produced 1O2 and O2-., although at varying degrees. Psoralen and 3-carbethoxypsoralen produced 1O2 greater than isopsoralens (angelicins). However, nonphotosensitizing angelicin, 5-methylangelicin, and 4,8-dimethyl-5'-carboxypsoralen produced 1O2 greater than 8-methoxypsoralen and 5-methoxypsoralen. The three monofunctional angelicin derivatives (isopsoralens) produced more O2-. than 8-methoxypsoralen, 5-methoxypsoralen, and 3,4'-dimethyl-8-methoxypsoralen. 3-Carbethoxypsoralen, a potent generator of 1O2 and a moderate producer of O2-., was highly photolabile. Until recently, skin photosensitization reactions (erythema, edema, damage to DNA or the membrane of cutaneous cells, the inhibition of scheduled DNA synthesis and skin carcinogenesis, etc.) were believed to involve photocyclo-addition of psoralens to DNA mediated by a type-I or anoxic reaction (a sensitizer-substrate interaction through the transfer of hydrogen atoms or electrons, but no direct involvement of molecular oxygen). Oxygen-dependent sensitized photodynamic reactions of type-II, involving the production of reactive oxygen (1O2 and O2-.), were believed not to mediate psoralen photosensitization reactions. We suggest that 1O2 and O2-. may also participate in skin photosensitization and cell membrane-damaging reactions. The fact that certain monofunctional isopsoralens produce 1O2 and O2-. at rates comparable to or better than bifunctional psoralens suggests that these reactive moieties of oxygen could play a major role in explaining their recently observed carcinogenic property and cell membrane-damaging reactions (e.g., edema or inflammation, etc.).     

Mutational specificity of gamma-radiation-induced guanine-thymine and thymine-guanine intrastrand cross-links in mammalian cells and translesion synthesis past the guanine-thymine lesion by human DNA polymerase eta

Comparative mutagenesis of gamma- or X-ray-induced tandem DNA lesions G[8,5-Me]T and T[5-Me,8]G intrastrand cross-links was investigated in simian (COS-7) and human embryonic (293T) kidney cells. For G[8,5-Me]T in 293T cells, 5.8% of progeny contained targeted base substitutions, whereas 10.0% showed semitargeted single-base substitutions. Of the targeted mutations, the G --> T mutation occurred with the highest frequency. The semitargeted mutations were detected up to two bases 5' and three bases 3' to the cross-link. The most prevalent semitargeted mutation was a C --> T transition immediately 5' to the G[8,5-Me]T cross-link. Frameshifts (4.6%) (mostly small deletions) and multiple-base substitutions (2.7%) also were detected. For the T[5-Me,8]G cross-link, a similar pattern of mutations was noted, but the mutational frequency was significantly higher than that of G[8,5-Me]T. Both targeted and semitargeted mutations occurred with a frequency of approximately 16%, and both included a dominant G --> T transversion. As in 293T cells, more than twice as many targeted mutations in COS cells occurred in T[5-Me,8]G (11.4%) as in G[8,5-Me]T (4.7%). Also, the level of semitargeted single-base substitutions 5' to the lesion was increased and 3' to the lesion decreased in T[5-Me,8]G relative to G[8,5-Me]T in COS cells. It appeared that the majority of the base substitutions at or near the cross-links resulted from incorporation of dAMP opposite the template base, in agreement with the so-called "A-rule". To determine if human polymerase eta (hpol eta) might be involved in the mutagenic bypass, an in vitro bypass study of G[8,5-Me]T in the same sequence was carried out, which showed that hpol eta can bypass the cross-link incorporating the correct dNMP opposite each cross-linked base. For G[8,5-Me]T, nucleotide incorporation by hpol eta was significantly different from that by yeast pol eta in that the latter was more error-prone opposite the cross-linked Gua. The incorporation of the correct nucleotide, dAMP, by hpol eta opposite cross-linked T was 3-5-fold more efficient than that of a wrong nucleotide, whereas incorporation of dCMP opposite the cross-linked G was 10-fold more efficient than that with dTMP. Therefore, the nucleotide incorporation pattern by hpol eta was not consistent with the observed cellular mutations. Nevertheless, at and near the lesion, hpol eta was more error-prone compared to a control template. The in vitro data suggest that translesion synthesis by another Y-family DNA polymerase and/or flawed participation of an accessory protein is a more likely scenario in the mutagenesis of these lesions in mammalian cells. However, hpol eta may play a role in correct bypass of the cross-links.     

Interstrand cross-links and sequence specificity in the reaction of cis-dichloro(ethylenediamine)platinum(II) with DNA

Characterization of the adducts produced in DNA by the cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) and a radiolabeled analogue, [3H]-cis-dichloro(ethylenediamine)platinum(II) ([3H]-cis-DEP) was recently reported [Eastman, A. (1983) Biochemistry 22, 3927]. Both drugs reacted at identical sites in DNA, most of which produced intrastrand cross-links. DNA-interstrand cross-links, which represent less than 1% of total platination, have now been characterized. DNA containing interstrand cross-links was enriched for on the basis of its renaturability after boiling. This DNA was digested to deoxyribonucleosides, and the adducts were separated by high-pressure liquid chromatography. A cross-link between two deoxyguanosines was observed to be the most prominent adduct. It is proposed that the major sequence in which this cross-link occurs is 5'-CG-3'. DNA that was incubated with [3H]-cis-DEP for 1 h showed low levels of interstrand cross-links. After removal of unreacted drug, their frequency increased significantly over 6 h with a maximum occurring at about 12 h. A similar phenomenon was seen in the case of intrastrand cross-links that contained adenine, in particular when the cross-link was between the terminal bases in an ANG trinucleotide sequence (N is any nucleotide). The primary site of reaction is at guanine, with a slow subsequent cross-link to the adenine. A model is presented that is consistent with the observation that adenine is always at the 5' terminus of these adducts. The proportion of adducts at ANG sequences also increased at elevated temperatures. This is discussed with regard to potential significance during hyperthermia treatment of patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA-protein cross-linking between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured human cells.

Formation of DNA-protein cross-links between thymine and tyrosine in chromatin of gamma-irradiated or H2O2-treated cultured human cells is reported. Chromatin was isolated from cells, and subsequently hydrolyzed and derivatized. Analysis of derivatized hydrolysates by gas chromatography/mass spectrometry with selected-ion monitoring showed that 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)-methyl]-L-tyrosine (Thy-Tyr cross-link) was formed. The presence of this DNA-protein cross-link in control cells was also observed at a level of approximately 7 molecules per 10(6) DNA nucleotides. Exposure of cells to ionizing radiation at doses between 8.7 and 82 Gy (J.kg-1) increased the amount of the Thy-Tyr cross-link linearly up to approximately fourfold over the background level. At doses higher than 82 Gy, the yield approached a plateau. Treatment of cells with H2O2 (0.5 to 10 mM) also increased the amount of the Thy-Tyr cross-link in a concentration-dependent manner. Addition of dimethyl sulfoxide and o-phenanthroline in the culture medium afforded partial inhibition of cross-link formation. Addition of catalase inhibitor KCN prior to H2O2 treatment increased the yield of cross-linking over the level observed with H2O2 treatment alone. Pretreatment of cells with ascorbic acid for 24 h without H2O2 caused formation of the Thy-Tyr cross-link. This DNA-protein cross-link in chromatin of cells is proposed to be formed by mechanisms involving a radical addition reaction and/or a radical-radical combination involving thymine and tyrosine radicals. Hydroxyl radical mediated by chromatin-bound metal ions is proposed to cause the formation of the Thy-Tyr cross-link in H2O2-treated cells.     

The mechanisms of action of phototherapy in the treatment of the most common dermatoses

Phototherapy denotes the use of ultraviolet (UV) light in the management of several dermatoses. Most phototherapy regimens utilize ultraviolet radiation of different wavelenghts. Currently, irradiations with broadband UVB (290-320 nm), narrowband UVB (311-313 nm), 308 nm excimer laser, UVA 1 (340-400 nm), UVA with psoralen (PUVA), and extracorporeal photochemotherapy (photopheresis) are being used. The interplay of the various photobiologic pathways is far from being completely understood. Disordes that may benefit from such approach are numerous, with psoriasis, atopic dermatitis, cutaneous T-cell lymphomas, morphea, and vitiligo as main indications. The immunomodulatory effects of UVB radiation primarily affect the epidermis and superficial dermis, while UVA radiation affects mid and deep dermal components, especially blood vessels. UVB radiation is absorbed by endogenous chromophores, such as nuclear DNA, which initiates a cascade of events. Absorption of UV light by nucleotides causes the formation of DNA photoproducts and suppresses DNA synthesis. In addition UV light stimulates synthesis of prostaglandins and cytokines that play important roles in immune suppression. It may reduce the number of Langerhans cells, cutaneous T lymphocytes and mast cells in the dermis. UV radiation can also affect extranuclear molecular targets located in the cytoplasm and cell membrane. Immune suppression, alteration in cytokine expression, and cell cycle arrest may all contribute to the suppression of disease activity. PUVA is a form of chemophototherapy which uses UVA light to activate chemicals known as psoralens, hence psoralen ultraviolet A. The conjunction of psoralens with epidermal DNA inhibits DNA replication and causes cell cycle arrest. Psoralen photosensitization also causes an alteration in the expression of cytokines and cytokine receptors. Psoralens interact with RNA, proteins and other cellular components and indirectly modify proteins and lipids via singlet oxygen-mediated reactions or by generating of free radicals. Infiltrating lymphocytes are strongly suppressed by PUVA, with variable effects on different T-cell subsets. Psoralens and UV radiation also stimulate melanogenesis. Extracorporeal photopheresis is technique used in treatment of erythrodermic cutaneous lymphomas. It is very potent in induction of lymphocyte apoptosis. Despite the introduction of numerous effective systemic medications and biologic agents in dermatology, phototherapy remains a reliable, and often preferred option for several dermatoses.