Paracetamol inhibits UV-induced DNA repair in resting human mononuclear blood cells in vitro

The effects of paracetamol on the repair of DNA damage in restinghuman peripheral mononuclear blood cells (MNC) in vitro wereinvestigated by means of the alkaline elution technique. Lowdoses of UV light (254 nm, 3 J/m²) caused a transient increasein the amount of DNA singlestrand breaks and alkali-labile sites(SSBs). Paracetamol (0.1–1.0 mM) present during post-irradiationincubation approximately doubled the maximum level of UV-induced(1–3 J/m²) SSBs and delayed the completion of repair.Although there were considerable variations between cells preparedfrom different donors, the level of UV-induced DNA SSBs wasalways higher with paracetamol. Hydroxyurea (0.3 mM), an inhibitorof ribonucleotide reductase, caused a similar increased accumulationand slow removal of SSBs, whereas cytosine-l-ß-D-arabinofuranoside(Ara C) (10 µM), an inhibitor of DNA polymerases, ledto a steady accumulation of DNA SSBs. The increased levels ofSSBs caused by paracetamol or hydroxyurea were both completelysuppressed by concomitant addition of deoxyribonucleosides;this supports the notion that paracetamol as well as hydroxyureainhibits ribonucleotide reductase. About the same rates of formationand removal of UV-induced SSBs were observed in T lymphocytes,B lymphocytes and monocytes. In both isolated T lymphocytesand B lymphocytes, paracetamol (0.3 mM) markedly increased thelevel of DNA SSBs induced by UV, whereas monocytes seemed tobe less sensitive to the effect of paracetamol. It is concludedthat the inhibition of DNA repair may contribute to the clastogeniceffects of paracetamol.     

Damage proneness induced by genomic DNA demethylation in mammalian cells cultivated in vitro

Variations in the genomic DNA methylation level have been shownto be an epigenetic inheritable modification affecting, amongother targets, the sister chromatid exchange (SCE) rate in mammaliancells in vitro. The inheritable increase in SCE rate in affectedcell populations appears as a puzzling phenomenon in view ofthe well established relation between SCE and both mutagenesisand carcinogenesis. In the present work we demonstrate that,in a treated cell population, demethylation could be responsiblefor the inheritable induction of damage proneness affectingboth damage induction and repair. Normal and ethionine or azacytidinetreated Chinese hamster ovary cells, subclone K1 (CHO-K1), werechallenged with UV light (UV) or mitomycin-C (MMC) at differenttimes from the demethylating treatment. The SCE rate was measuredwith two main objects in view: (i) the induction of synergismor additivity in combined treatments, where mutagen (UV or MMC)pulse is supplied from 0 to 48 h after the end of the demethylatingtreatment; and (ii) the pattern of damage extinction, for theduration of up to six cell cycles after the end of the combined(demethylating agent + mutagen) treatment. Results indicateboth a synergism in SCE induction by mutagens in demethylatedcells even if supplied up to four cell cycles after the endof the demethylation treatment and a delay in recovery of induceddamage, compared with normally methylated cells. These dataare discussed in the light of the supposed mechanism of SCEincrease and of the possible biological significance in termsof mutagenesis and carcinogenesis. ³To whom correspondence should be addressed     

Role of DNA repair by (A)BC excinuclease and Ogt alkyltransferase in the final distribution of LacI-d mutations induced by N-butyl-N-nitrosourea in Escherichia coli

In the absence of nucleotide excision repair, the additionaldeficiency of the DNA alkyltransferase (ATase) encoded by theconstitutive ogt gene of Escherichia coli caused a marked increasein mutation induction by N-butyl-N-nitrosourea (BNU). Irrespectiveof the presence or absence of the Ogt ATase, little mutagenicresponse was detected in Uvr⁺ bacteria in the concentrationrange 0–8 mM BNU, indicating that most premutagenic DNAlesions induced at these concentrations are efficiently recognizedand repaired by the nucleotide excision repair system. Increasedsusceptibility to mutagenesis by BNU was detected in Uvr^–Ogt⁺ bacteria, but the Uvr^– Ogt^– double mutant exhibitedmuch higher sensitivity. These data suggest that the Ogt ATasecan replace to a great extent the repair capacity of the (A)BCexcinuclease. Forward mutations induced by 6 mM BNU within theinitial part of the lacI gene of E.coli were recovered fromUvr⁺ Ogt^–, Uvr^– Ogt⁺ and Uvr^– Ogt^– bacteria.A total of 454 independent mutations were characterized by DNAsequence analysis. The BNU-induced spectra were dominated byG:C     

Differential effects of cordycepin on the induction of sister-chromatid exchange and chromatid breaks in BALB/Mo mouse lymphocytes treated with mitomycin C

BALB/Mo mice lymphocytes, carrying endogenous Moloney murineleukaemia virus (M-MuLV), show significantly higher in vitrobaseline frequencies of sister chromatid exchange (SCE) thanlymphocytes from control (M-MuLV free) BALB/c mice. In vitrotreatment of lymphocytes with cordycepin (10 µg/ml), anantiviral substance, lowers the level of SCE in BALB/Mo cellsto the same value of BALB/c cells. The drug also reduces thehigher sensitivity of BALB/Mo compared to BALB/c lymphocytesto the induction of SCE by mitomycin C (MMC) administered eitherin vitro (3 x 10^–8, 10^–7 M) or in vivo (0.3, 3 mg/kg).BALB/Mo lymphocytes treated in vivo with a high dose of MMC(10 mg/kg) show reduced susceptibility to the induction of SCEbut increased frequencies of chromatid breaks and micronuclei.In this situation, cordycepin increases the level of SCE inBALB/Mo lymphocytes to exactly the level seen in BALB/c cells,but it does not affect the frequency of chromosomal aberrations.Since cordycepin is known to inhibit poly(A) synthesis, thusblocking RNA maturation, it is suggested that M-MuLV proviralintegration is not the sole factor responsible for the alteredsusceptibility of BALB/Mo lymphocytes to SCE induction, butthat it is most likely viral gene expression that is neededfor this effect to occur. On the contrary, the high susceptibilityof BALB/Mo lymphocytes to the induction of chromatid aberrationsby a high dose of MMC administered in vivo seems to be independentof viral maturation. ²To whom correspondence should be addressed     

Induction of SCE by indirect mutagens in cultured rat hepatoma cells and in Chinese hamster V79 cells co-cultivated with hepatocyte primary cultures

The continuous rat hepatoma cell line H4IIEC3/G^– and rathepatocyte primary cultures (hpc) were compared with regardto their capacity to metabolize structurally different promutagens.The sensitivities of both activation systems were evaluatedby comparing the induction of SCE in H4IIEC3/G^– cellsthemselves with that in V79 cells co-cultured with hpc. Of thesix chemicals tested, aflatoxui B₁ (AFB1), cyclo-phosphamide,dimethylnitrosamine and nitrosomorpholine (NM) were shown tobe inducers of SCE in H4HEC3/G^– cells as well as in V79cells with hepatocyte activation. 7,12-Dimethylbenzanthracenegave positive responses in hpc/V79 co-cultures but not in H4IIEC3/G^–cells whereas benzo[a]pyrene was negative in both systems. Theseresults suggest that H4IIEC3/G^– cells retain metabolicactivities to convert different indirect mutagens into theiractive forms and clearly indicate the presence of liver specificcytochrome P-450-dependent mono-oxygenases. However, freshlyisolated hepatocytes are more efficient in metabolizing thetest compounds. Although hpc provide only external activation,the V79 cells system appears to be more sensitive for the detectionof promutagens.     

Uptake and DNA photodamage induced in plant cells in vivo by two cationic porphyrins

The in vivo uptake of two cationic porphyrins: mesotetra (4-N-methylpyridyl)porphine (T₄MPyP) and its zinc complex (ZnT₄MPyP) was determinedin Allium cepa meristematic cells. Both photosensitizers (10^–7M for 4 h) penetrated into the nucleus producing a red fluorescenceof chromatin under blue-violet (436 nm) exciting light. Theability of T₄MPyP and ZnT₄MPyP to induce DNA photodamage wasmeasured by the sister chromatid exchange (SCE) test. 5-Bromo-2'-deoxyuridine-substitutedchromosomes treated with both the porphyrins (10^–8 M for4 h) showed increased frequencies of SCE when they were postirradiatedwith 436 nm light. A higher genotoxic effect was observed forZnT₄MPyP than the other compound. ¹To whom correspondence should be addressed     

A single-site mutation in the XPAC gene alters photoproduct recognition

The XPAC (xerodenna pigmentosum group A complementing) gene,which is located on chromosome 9, carries a variety of pointmutations in XP group A patients. We investigated the role ofthe XPAC gene product in excision repair by generating revertantsof an XP group A cell line (XP12RO) that have increased resistanceto ultraviolet light. One of these cell lines, XP129, can repair(6–4) pyrimidine-pyrimidone photoproducts normally buthas reduced repair of cyclobutane dimers, as in XP12RO. Sequenceanalysis of cDNA from the XPAC gene indicated that XP12RO containsa termination codon at amino acid position 207, resulting ina reduced amount of mRNA and no detectable protein. In the revertantXP129 line, this termination codon has been mutated furtherand now encodes giycine in one allele instead of the wild-typearginine. The mRNA level detected by allelespecific polymerasechain reaction amplification was greater for the reverted sequencethan for the chain-terminating sequence. These observationsindicated that a point mutation resulting in a mis-sense mutationin the XPAC gene and altered expression of the XPAC proteincan alter the substrate specificity of the excision repair system,and imply that the XPAC gene product plays an important rolein photoproduct recognition. ¹To whom correspondence should be sent     

Regional mapping of human DNA excision repair gene ERCC4 to chromosome 16pl3.13-pl3.2

Mitomycin C (MMC)-resistant interspecific somatic cell hybridsmade between human cells and the MMC-sensitive, Chinese hamsterovary (CHO) excision repair-deficient UV41 cells generally containedhuman chromosome 16, while other human chromosomes were randomlypresent. MMC-sensitive and -resistant subclones were isolatedfrom resistant clones and resistance generally segregated concordantlywith human chromosome 16 markers. UV radiation survival analysisof subclones indicated that MMC and UV resistance were correlated.Therefore the complementing gene, Excision Repair Cross Complementing4 (ERCC4) was assigned to human chromosome 16. Complementationof UV41 by human cells derived from patients with xerodermapigmentosum groups A, C, D and F excluded ERCC4 from involvementin those disease syndromes. Resistant hybrids containing onlyportions of chromosome 16 were identified by the lack of concordanceof multiple chromosome 16 markers. When such hybrids were usedas a source of probe for fluorescent in situ hybridization ontonormal human metaphases, the only region of chromosome 16 identifiedas being consistently present was 16pl3.1–pl3.3. Geneticmarker analysis of informative hybrids with mapped probes refinedthe position of ERCC4 to 16pl3.13–pl3.2 and allowed thefollowing order of markers within the region to be established:pter–(PRM1 D16S215)–D16S213–D16S53 –(D16S214,ERCC4)–D16S3–D16S96–cen ⁴To whom correspondence should be addressed     

Caffeine effect on the mitotic delay induced by G2 treatment with UVC or mitomycin C

It is well established that DNA lesions trigger cell cycle check-pointscausing a mitotic delay that is required for their repair beforecells enter the mitotic phase. Caffeine, in some cases, canremove this delay and consequently potentiates the yield ofinduced chromosome aberrations. The objective of this studywas to test the effect of a G2 treatment with S-dependent agents(UV light and mitomycin C) on the cell kinetics of a G2 cellpopulation and evaluate whether post-treatments with caffeinecould modulate removal of the expected cell cycle delay. Cellkinetics were monitored by analysing the mitotic index (MI)values in combination with the 5-bromo-2'-deoxyuridine (BrdUrd)labelling technique. Chinese hamster fibroblast cultures (AA8)were treated in G2 phase of the cell cycle with 8 and 15 J/m²UV light or 0.1 and 0.6 µg/ml mitomycin C for 1.5 h. Post-treatmentswith caffeine were performed at dose levels and recovery timeswhere the mitotic indices were substantially reduced. The resultsobtained showed that both UV light and mitomycin C induced aG2 arrest, as indicated by MI values and the absence of Brd-Urdlabelledmetaphases. For UV light the G2 block was observed at lowerand higher dose levels after 1.5 h, while for mitomycin C itwas observed only at the higher dose level after 1 h. However,in both cases the block lasted {small tilde}1 h, after which,even though slowed down, the cell population entered mitosis,as indicated by increased MI values. This block was not removedby caffeine post-treatment. In contrast, caffeine G2 post-treatmentwas able to remove G2 arrest induced by G1–treatments.Accordingly, our results suggest that both UV light- and mitomycinC-induced damage must be processed during S phase to allow caffeineto remove induced G2 blocks.      

A deficiency for nucleotide excision repair strongly potentiates the mutagenic effectiveness of methyl bromide in Drosophila

A DNA repair assay measuring hypermutability response in theabsence of nucleotide excision repair (NER) was employed tostudy the impact of a deficiency in NER on the induction offorward mutations (X-chromosomal recessive lethals) by methylbromide (MeBr) in Drosophila melanogaster. Postmeiotic malegerm-cell stages reacted with MeBr were introduced in eitherNER competent oocytes (exr⁺) or in cells from the NER^–strain mus-201. The high average M_exr^–/M_exr⁺ hypemutabilityratio of 8.3 determined for MeBr is similar to the M_exr^–/M_exr⁺indices found for other monofunctional alkylating agents withhigh Swain—Scott s values, such as methyl methanesulphonateand dimethyl sulphate. It is concluded that the genotoxic profileof methyl bromide is in keeping with those of high s-value alkylatingagents but it seems incompatible with a methylating agent producingsubstantial amounts of O⁶methylguanine. ¹To whom correspondence should be addressed     

Enhanced unscheduled DNA synthesis by secondary cultures of lung cells established from calorically restricted aged rats.

Unscheduled DNA synthesis (UDS) induced by two exposure levels of ultraviolet light (UV) or two concentrations of methyl methane sulfonate (MMS) was evaluated in secondary cultures of lung fibroblasts established from weanling, 11-month-old and 31-month-old female Fischer 344 rats fed ad libitum (AL) or calorically restricted (CR) diets. [3H]Thymidine incorporation as a function of UDS was highest for weanling-derived cells treated with either UV or MMS, declining consistently with increased age between cells from weanling, 11-month-old and 31-month-old animals. [3H]Thymidine incorporation as a function of UDS in cells from 11-month-old AL vs. CR rats differed only at the highest UV exposure level. In contrast, cells derived from 31-month-old CR rats exhibited UDS levels which were at least twice as high at each UV treatment level as UDS levels of cells derived from the same age AL rats. Cells from both old AL and old CR rats were shown to initiate DNA excision repair at about the same rate. Cells from CR rats, however, repaired DNA damage at an accelerated rate and completed excision repair while repair in cells from AL animals was slower and apparently did not proceed to completion. Data from this study indicate that cells from young and old AL and CR animals initiate excision repair, but demonstrate an age-related loss of UV- or MMS-stimulated [3H]thymidine incorporation in cells derived from AL animals. Cells derived from CR animals did not exhibit that age-related loss of UDS activity; rather, they showed an enhanced UDS response to DNA damage and appeared to complete ligation as the final step in excision repair. The data suggest that caloric restriction of a cell donor animal not only delays the age-associated decrease in in vitro DNA excision repair capacity in cells from that animal, but may actually enhance repair capacity.     

Repair of ultraviolet light damage in Saccharomyces cerevisiae as studied with double- and single-stranded incoming DNAs

Summary Purified double- and single-stranded DNAs of the autonomously replicating vector M13RK9-T were irradiated with ultraviolet light (UV) in vitro and introduced into competent whole cells of Saccharomyces cerevisiae. Incoming double-stranded DNA was more sensitive to UV in excision repair-deficient rad2-1 cells than in proficient repair RAD ⁺ cells, while single-stranded DNA exhibited high sensitivity in both host cells. The results indicate that in yeast there is no effective rescue of UV-incoming single-stranded DNA by excision repair or other constitutive dark repair processes.     

DNA damage induced by UV light affects restriction endonuclease recognition sites: correlation between effects at chromosomal level and naked DNA

Cytogenetic and molecular analyses were performed in G₁ Chinesehamster ovary cells treated with 254 nm UV light, to test thehypothesis that UV may affect the recognition site of specificrestriction endonucleases (RE). Since shortwavelength UV lightinduces mainly cyclobutane dimers (CPD) at TT and CT sequences,RE were selected according to the presence or absence of thymineat the recognition site (Dral TTT/AAA, AluI AG/CT and HaeIIIGG/CC). A drastic reduction of DraI- and AluI-induced chromosometypeaberrations was found in cells pretreated with UV. Conversely,such a reduction was not observed with HaeIII. To better understandthis phenomenon, a molecular analysis was carried out at boththe genome level and at the hypoxanthine phosphoribosyl transferasegene level, showing that the cutting pattern of Dral on isolatedDNA from UV-irradiated cells was strongly reduced compared withan untreated sample, whereas HaeIII was not able to modify thecutting pattern of irradiated cells. Our data demonstrate agood correlation between the results obtained with cytogeneticand molecular approaches, suggesting that cyclobutane dimersare the main lesions responsible for the observed reductionof the cleaving activities of RE, at both the chromatin andnaked DNA levels. ⁵To whom correspondence should be addressed at: Dip. di Genetica e Biologia Molecolare, Università ‘La Sapienza’ P.le A. Moro 5, 00185-Roma, Italy