32P-Post-labeling detection of DNA adducts in mononuclear cells of workers occupationally exposed to styrene

³²-Post-labeling was used to analyze for the presence of DNAadducts in 47 workers exposed to styrene in a boat manufacturingfacility. Individual airborne exposures measured several timesover the course of 1 year ranged from 1to 235 mg/m³ with a meanvalue of 65.6 mg/m³. Two adducts were detected in the DNA ofmononuclear cells of these workers. The following levels ofadducts were detected: adduct 1, range 0.6–102x10^–8(mean 15.8x10^–8); adduct 2, range 0.1–70.9x10^–8(mean 14.2x10^–8). Significant linear relationships werefound between styrene exposure and both DNA adducts (adduct2, r = 0.330, P = 0.012; adduct 1, r = 0.244, P = 0.049). Co-chromatographyexperiments identified DNA adduct 1 in the exposed samplesasN²-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine-3', 5'-bisphosphate.DNA adduct 2 remains unidentified. No significant linear relationshipswere observed between the level of DNA adducts and sister chromatidexchanges, possibly because of the poor precision of the ³²-post-labelingassay (the estimated coefficients of variation for adducts 1and 2 were 2.54 and 1.96, respectively). These results demonstratethat occupational exposure to styrene results in the formationof DNA adducts in human mononuclear cells.     

DNA adduct formation in the bone marrow of B6C3F1 mice treated with benzene

We used P₁-enhanced ³²P-postlabeling to investigate DNA adductformation in the bone marrow of B6C3F1 mice treated intraperitoneallywith benzene (BZ). No adducts were detected in the bone marrowof controls or mice treated with various doses of BZ once aday. After twice-daily treatment with BZ, 440 mg/kg, for 1 to7 days, one major and two minor DNA adducts were detected. Therelative adduct levels ranged from 0.06–1.46x10^–7.In vitro treatment of bone marrow from B6C3F1 mice with variousdoses of hydroquinone (HQ) for 24 h also produced three DNAadducts. These adducts were the same as those formed after invivo treatment of bone marrow with BZ. Co-chromatography experimentsindicated that the principal DNA adduct detected in the bonemarrow of B6C3F1 mice was the same as that detected in HL-60cells treated with HQ. This finding suggests that HQ may bethe principal metabolite of BZ leading to DNA adduct formationin vivo. DNA adduct 2 corresponds to the DNA adduct formed inHL-60 cells treated with 1,2,4-benzenetriol. DNA adduct 3 remainsunidentified. After a 7-day treatment with BZ, 440 mg/kg twicea day, the number of cells per femur decreased from 1.6x10⁷to 0.85xlO⁷, indicating myelotoxicity. In contrast, administrationof BZ once a day produced only a small decrease in bone marrowcellularity. These studies demonstrate that metabolic activationof BZ leads to the formation of DNA adducts in the bone marrow.Further investigation is required to determine the role of DNAadducts and other forms of DNA damage in the myelotoxic effectsof exposure to BZ.     

SHORT COMMUNICATION: Detection of DNA adducts in the white blood cells of B6C3F1 mice treated with benzene

We have employed the P₁-enhanced ³²P-postlabeling procedureto detect the formation DNA of adducts in the white blood cells(WBC) of B6C3F1 mice treated by i.p. injection with benzene.Treatment twice a day with 440 mg/kg benzene for 1–7 daysresulted in the formation of one major (adduct 1) and one minor(adduct 2) DNA adduct in the WBCs of mice. The same DNA adductpattern was also found in the bone marrow (BM) of benzene treatedmice. The relative adduct levels were dependent upon both benzenedose from 100–440 mg/kg and treatment time from 1 to 7days. The relative adduct levels ranged between 0.11 and 1.33adducts in 10⁷ nucleotides for WBCs and 0.16–1.21 adductsin 107 nucleotides for BM. Following treatment with benzene,the levels of DNA adducts formed in WBCs were significantlycorrelated with the levels of DNA adducts formed in BM (r² =0.97, P <0.001). Our results suggest that measurement ofDNA adducts in WBCs may be an indicator of DNA adduct formationin BM following BZ exposure.     

Activation of 4-hydroxytamoxifen and the tamoxifen derivative metabolite E by uterine peroxidase to form DNA adducts: Comparison with DNA adducts formed in the uterus of Sprague-Dawley rats treated with tamoxifen

Daily intraperitoneal treatment of female Sprague-Dawley ratswith either 5, 10 or 20 mg/kg tamoxifen (TAM) for 1 weeks increasedthe level of peroxidase activity in the uterus 2- to 10-foldcompared to the control level. Using uterine extracts preparedfrom control and TAM treated animals, we investigated the activationof 4-hydroxytamoxifen (4-HO-TAM) and (E, Z)-1, 2-dipheny-1-(4-hydroxyphenyl)-but-1-ene(cis/trans-metabolite E) to form DNA adducts. Activation of4-HO-TAM by uterine extracts prepared from either control orTAM-treated rats produced one major (a) and Two minor DNA (band c) adducts. A similar activation of cis/trans-metaboliteE produced two adducts (d and e). There was good correlationbetween levels of uterine peroxidase activity and levels ofDNA adducts formed by 4-HO-TAM and cis/trans-metabolite E. Activationof 4-HO-TAM and cis/trans-metabolite E with horseradish peroxidase(HRP) produced the same adducts as observed by activation withuterine extract Treatment of Sprague-Dawley rats with 5 and10 mg/kg for 7 days produced eleven DNA adducts in the liverwith no adducts detected in the uterus. However, treatment ofrats with 20 mg/kg of TAM for 7 days produced the same adductpattern in the liver and also one major adduct (1) in the uteruswith a relative adduct level of 6.4 ±4.1x10^–9.Tamoxifen-DNA adduct 1 detected both in the liver and in theuterus of treated rats was similar to adducts produced by activationof 4-HO-TAM with either uterine extract or HRP. The resultsof these studies suggest a general model whereby the tamoxifenmetabolite 4-HO-TAM is further activated in the uterus by peroxidaseenzymes to form DNA adducts.     

Mutagenicity of butadiene and its epoxide metabolites: II. Mutational spectra of butadiene, 1,2-epoxybutene and diepoxybutane at the hprt locus in splenic T cells from exposed B6C3F1 mice

The mutagenic potential and mutational spectra of butadiene(BD), 1,2-epoxybutene (EB), and diepoxybutane (DEB) were determinedin splenic T cells from exposed B6C3F1 mice. Mice exposed byinhalation to 625 p.p.m. BD for 2 weeks displayed an averagehprt^– mutation frequency of 6.2 x10^–6 compared to1.2x10^–6 in controls. Mice were also given three dailyi.p. doses of 60, 80 and 100 mg EB/kg or 7, 14 and 21 mg DEB/kg.Average hprt^– frequencies of 5.4x10^–6, 4.lx10^–6and 8.6x10^–6 were seen in the EB groups, respectively,while average frequencies of 4.6x10^–6, 9.4x10^–6and 13x10^–6 were seen in the DEB groups. DNA sequencingrevealed that approximately half of the mutations induced invivo by BD, EB and DEB were frameshift mutations. A +1 frameshift‘hotspot’ in six consecutive guanine bases in exon3 was observed with all three compounds. The remaining mutationsproduced by BD, EB and DEB were transition and transversionmutations at both AT and GC base pairs. Base pair substitutionsinduced by BD were biased in favor of mutation at AT base pairs.The mutational spectra produced by BD, EB and DEB were verysimilar to that observed previously with ethylene oxide, suggestingthat these epoxide agents may be working through a similar mutagenicmechanism.     

In vitro binding of aflatoxin B1 and 2-acetylaminofluorene to rat, mouse and human hepatocyte DNA: the relationship of DNA binding to carcinogenicity

DNA binding levels were determined and compared in culturedhepatocytes from male and female rats as well as other animalspecies following exposure to aflatoxin B₁ (AFB₁) or 2-acetylaminofluorene(2-AAF). When human, rat (both male and female) and mouse hepatocytesin primary culture were exposed to 2.0 ? 10^–7 M [³H]AFB₁(sp. act. 2.63 µCi/nmol) for 24 h, male rat hepatocyteshad the highest degree of [³H]AFB₁-DNA binding (203 pmol/mgDNA) and human hepatocytes contained the next highest bindinglevel (42 pmol/mg DNA). Hepatocytes from female rats contained38 pmol/mg DNA while cultured mouse hepatocytes contained only1.4 pmol/mg DNA. When the same dose of [³H]AFB₁ was administeredto the cultured male rat hepatocytes at 24 h, 48 h, 72 h and1 week after seeding, and incubated for 24 h, the DNA bindinglevels were 189, 175, 76, 75 pmol/mg DNA respectively. In parallelexperiments to the cultured male rat hepatocytes above, theAFB₁-DNA binding levels in the cultured female hepatocytes were42, 41, 37 and 34 pmol/mg DNA respectively. Human, male andfemale rat hepatocytes in primary culture were exposed to 5.2? 10^–5 M 2-acetyl-amino [9–¹⁴C]fluorene (sp. act.0.0094 µCi/nmol) for 24 h. It was determined that malerat hepatocytes contained the highest amount of radioactivelylabeled 2-AAF bound to their DNA (1.57 nmol/mg DNA), femalerat hepatocytes contained 0.62 nmol/mg DNA and human hepatocytescontained 0.29 nmol/mg DNA. Results from our in vitro hepatocyteculture system correlate well with in vivo animal studies dealingwith species and sex differences in DNA binding and carcinogenicsusceptability. This indicates that hepatocytes in vitro maintainmany of the biological properties necessary for carcinogen responsesimilar to liver cells in vivo. In addition, comparison of genotoxiceffect in cultured hepatocytes from animals as well as humansmay be useful in evaluating carcinogenic potential of xenobioticsin human liver.     

DNA adduct formation and assessment of aberrant crypt foci in vivo in the rat colon mucosa after treatment with N-methyl-N-nitrosourea

N-Nitroso-compound DNA adduct formation in vivo and occurrenceof aberrant crypt foci (ACF) were studied in the rat colon mucosaafter a single, local treatment with a carcinogen, N-methyl-N-nitrosourea(MNU), using a simple surgical approach. A segment of F344 ratcolon was ligated to make a pouch and injected with MNU solution.For the study of DNA adduct formation, the solution contained50 µCi of [³H]MNU. The results demonstrated that similarranges of carcinogen dose, i.e. 0.15x10^–2 –1.5x10^–2MMNU, could induce both DNA adduct formation and appearanceof ACF in the rat colon with both parameters showing a nearbylinear dose dependence. HPLC analysis revealed the DNA adductsto include both 7-methylguanine (7-mGua) and O⁶-methylguanine(O⁶-mGua) with the 7-mGua/O⁶-mGua ratio being 8.2–11.3:1in the system used. Assessment of ACF development from 4 to16 weeks after MNU treatment at a dose of 7.5x10^–2 M showedthe numbers to increase up to the 8th week, followed by a decreaseat weeks 12 and 16, when 40% of the ACF counted at the peaktime point were still present. The percentage of large ACF (     

Carcinogen-mediated induction of SV40 DNA amplification is enhanced by acrylamide in Chinese hamster CO60 cells

The exposure of SV40-transformed Chinese hamster cells (lineCO60) to 50–150 µg/ml of monomeric acrylamide for24 h resulted in a very weak induction of the amplificationof the SV40 DNA inserts as measured by in situ hybridizationwith radioactive SV40 DNA as probe. The weak SV40 DNA amplificationobserved might result from a weak DNA-damaging activity as biologicallyillustrated by the capacity of high concentrations of acrylamideto irreversibly inhibit the DNA synthesis rate of CO60 cells.Moreover, acrylamide synergistically enhanced both the cytotoxicityand the induction of SV40 DNA synthesis by the carcinogens ethylmethanesulfonate, benzo[a]pyrene, mitomycin C, 4-nitroquinoline-l-oxide,8-azaguanine and 5-fluorodeoxyuridine. Although a weak inducerof SV40 DNA amplification by itself, acrylamide potentiatedthe genotoxicity of a series of chemical carcinogens. This findingshould be taken into consideration when assessing the risk ofthis widely used chemical.     

Pretreatment of normal human fibroblasts and human colon carcinoma cells with MNNG allows chloroethylnitrosourea to produce DNA interstrand crosslinks not observed in cells treated with chloroethylnitrosourea alone

Chloroethylnitrosoureas (CNU) are antitumor agents which produceDNA interstrand crosslinks. We have proposed that crosslinksare produced in DNA via monoadduct formation at the guanine-O⁶position, followed by a delayed reaction with the opposite DNAstrand. Human cells are known to differ in their capacity torepair the O⁶-methylguanine lesion. One example of this repaircapacity is the ability of cells to reactivate adenovirus whichhas been damaged by in vitro treatment with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG). Cells that repair the virus are designated Mer⁺ anddeficient cells Mer^–. In a recent report, we showed aclear correlation between CNU-induced DNA interstrand cross-linkingand the Mer phenotype. Mer^– cells produced consistentlyhigher levels of interstrand crosslinks than did Mer⁺ cells.In the present study we have measured the CNU-induced DNA interstrandcrosslinking in IMR-90 normal human fibroblasts (Mer⁺), HT-29human colon carcinoma cells (Mer⁺), and VA-13 SV-40 transformedhuman cells (Mer^–) following pretreatment with MNNG. Cellswere treated for 1 h with MNNG, then for an additional 1 h withCNU. Comparable levels of CNU-induced DNA interstrand crosslinkingwere observed in all cell lines. This crosslinking has beenpreviously undetected in the IMR-90 and HT-29 cells. Cytotoxicitystudies showed that MNNG pretreatment greatly enhanced the killingof IMR-90 and HT-29 cells by CNU, however, in VA-13 cells theincrease in cell kill was smaller. These data suggest that inMer⁺ cells a DNA repair system may remove chloroethyl monoadductsbefore the lethal DNA interstrand crosslinks can form. However,pretreatment of cells with MNNG may saturate this repair systemrendering it inoperable.     

DHFR gene amplification in cultured skin fibroblasts of ataxia telangiectasia patients after methotrexate selection

During selection for methotrexate resistance, SV40-transformedhuman skin fibroblasts from patients with ataxia telangiectasia(A-T) underwent amplification of the dihydrofolate reductase(DHFR) gene, experienced nearly complete loss of the integratedSV40 sequences and showed a 3.6-fold increase in Ki-ras genecopy number. Over a period of months methotrexate-resistant(MTX^r) A-T subclones were obtained, which were able to growin progressively increasing MTX concentrations up to 100 µM.The ED₅₀ values determined as the effective dose of MTX causing50% growth inhibition in comparison to control cells increasedfrom 3x10^–2 µM for MTX^s AT5BI-VA cells to 250 µMMTX for the MTX^r AX100 subclone. In contrast, human skin fibroblastsof healthy individuals did not show DHFR gene amplificationand loss of SV40 sequences under comparable conditions and wereunable to grow in MTX concentrations >1 µM. Gene amplificationand loss of DNA sequences are features underlying the genomicinstability known to be a characteristic property of A-T cellsand being probably responsible for the high cancer incidencein these patients.     

Appearance of artefacts when using 32P-postlabelling to investigate DNA adduct formation by bile acids in vitro: lack of evidence for covalent binding

We reported (Scates et al. Carcinogenesis 1994, 15, 2945–2948)that incubating a range of bile acids with DNA in vitro, withor without exogenous metabolic activation, gave no evidenceof DNA adduct formation as judged by the nuclease P1 methodof ³²P-postlabelling. In contrast Hamada et al. (Carcinogenesis1994, 15, 1911–1915), also using postlabelling, claimedthat chenodeoxycholic acid, lithocholic acid, glycolithocholicacid and taurolithocholic acid bound covalently to DNA in vitro.To investigate this discordance we incubated solutions of salmonsperm DNA for 1 h at 37°C with 1 mg/ml of cholic acid, chenodeoxycholicacid, lithocholic acid, glycolithocholic acid or taurolithocholicacid. Each incubate was extracted extensively with diethyl etherafter which a sample of DNA was taken and ³²P-postlabelled usingthe nuclease P1 method. The DNA in the remaining incubate wasprecipitated from high salt solution with ethanol. Aliquotsof this DNA were postlabelled. The remainder of the DNA waspurified with proteinase-K, ribonuclease, phenol-chloroform,precipitated and postlabelled. Parallel incubates were madewith the same bile acids, under the same conditions but in theabsence of DNA and were then extracted, precipitated and postlabelledas described above. When DNA was present in the incubate butwas not precipitated, chenodeoxycholic acid, lithocholic acid,glycolithocholic acid and taurolithocholic acid, but not cholicacid, produced spots similar to those reported by Hamada etal. No such spots were seen when DNA was postlabelled afterprecipitation, or after precipitation and purification. Thesesame bile acids produced spots when postlabelled in the absenceof DNA, but spots were absent when these incubates were precipitatedand purified before postlabelling. We conclude that the spotsobtained when bile acids are incubated with DNA which is notprecipitated from high salt before it is postlabelled are technicalartefacts, and cannot be regarded as evidence that bile acidsbind covalently to DNA to form adducts. We also confirm reports(Vulimiri et al. Carcinogenesis 1994, 15, 2061–2064) thatbile acids alone can produce spots when incubated with T4 polynucleotidekinase and [     

Biomarkers of styrene exposure in lamination workers: levels of 06-guanine DNA adducts, DNA strand breaks and mutant frequencies in the hypoxanthine guanine phosphoribosyltransferase gene in T-lymphocytes

Occupational exposure to styrene was studied in nine workersof a hand lamination plant in Bohemia. Personal dosimeters wereused to monitor the styrene workplace exposure, and the levelsof styrene in blood and mandelic acid in urine were measured.Blood samples were taken at four occasions during a 7 monthperiod to determine styrene-specific 0⁶-guanine DNA adductsin lymphocytes and granulocytes, DNA strand breaks and hypoxanthineguanine phosphoribosyltransferase (HPRT) mutant frequency inT-lymphocytes. Seven administrative employees in the same factory(factory controls) and eight persons in a research laboratory(laboratory controls) were used as referents. DNA adduct levelsdetermined by the ³²P-postlabelling method in lymphocytes oflamina-tors were remarkably constant and significantly higher(P < 0.0001) than in factory controls at all four samplingtimes. HPRT mutant frequencies (MF) measured by the T-cell cloningassay were higher in the laminators (17.5 x10^–6, groupmean) than in the factory controls (15.7x10^–6, group mean)at three of the four sampling times, but the differences werenot statistically significant. However, a statistically significant(P = 0.021) difference between MF in the laminators (18.0 x10^–6,group mean) and laboratory controls (11.8 xl0^–6, groupmean) was observed at sampling time 4 (the only sampling timewhen this latter group was studied). This result indicates thatstyrene exposure may induce gene mutation in T-cells in vivo.DNA strand breaks were studied by the ‘Comet assay’at the fourth sampling time. The laminators were found to havesignificantly higher levels of DNA strand breaks than the factorycontrols (P = 0.032 for tail length, TL; P = 0.007 for percentageof DNA in tail, T%; and P = 0.020 for tail moment, TM). A statisticallysignificant correlation was also found between the levels oflymphocyte DNA adducts and all three DNA strand break parameters(TL P = 0.046; T% P = 0.026 and TM P = 0.034). On the contrary,no significant correlations were found between DNA adduct levelsand the HPRT mutant frequencies or between the mutant frequenciesand DNA strand breaks. Taken together, these results add furthersupport to the genotoxic and possibly mutagenic effects of styreneexposure in vivo. However, no simple quantitative relationshipseems to exist between the levels of styrene-induced DNA damageand frequency of HPRT mutation in T-lymphocytes.     

The binding efficiency of polyclonal and monoclonal antibodies to DNA modified with benzo[a]pyrene diol epoxide is dependent on the level of modification. Implications for quantitation of benzo[a]pyrene-DNA adducts in vivo

A number of polyclonal antibodies specific for DNA modifiedwith (±)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE) were obtained from the sera of New Zealand white rabbitsimmunized with BPDE-DNA, complexed with methylated bovine serumalbumin (mBSA). Monoclonal antibodies were developed by fusionof mouse myeloma cells with spleen cells isolated from BALB/cmice immunized with the same complex of BPDE-DNA and mBSA. Theseantibodies have been characterized for specificity in a highlysensitive, enzyme-linked immunosorbent assay (ELISA). All antibodiesshowed a very high affinity for single-stranded BPDE-DNA, buthad lower affinity towards native BPDE-DNA. The affinity forthe free mononucleoside BPDE-dG was at least 100-fold lowerthan that for BPDE-DNA, and no affinity was detected for BPtetrols or DNA modified with N-acetoxy-N-acetyl-2-aminofluorene.A high cross reactivity was observed with DNA modified with(±)-trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene.Using five different antibodies, monoclonal or polyclonal, weobserved that the antibody affinity for BPDE-DNA was dependenton the level of modification; in the competitive ELISA as littleas 4 fmol BPDE-DNA (50 pmol/µg) was sufficient for 50%inhibition with our best antisera, but 17 fmol of the adductwas required when [³H]BPDE-DNA of low modification (1–10fmol/µg) was used as inhibitor. When samples of [³H]BP-DNAisolated from the livers of mice, treated i.p. with differentdoses of [³H]BP were examined by competitive ELISA and calibratedwith [³H]BPDE-DNA of low modification (1–10 fmol/µg),binding values calculated from the immunoassay were in goodagreement with those obtained from radioactivity measurements.In contrast, when this DNA was quantitated in competitive ELISAusing highly modified BPDE-DNA as standards, values by ELISAwere 20–40% of those obtained by radioactivity. Theseresults indicate that the use of serially diluted BPDE-DNA ofhigh modification as standard competitor in the ELISA will leadto erroneous results in the measurement of adducts in DNAs modifiedto a low extent (biological samples). The property of antiseraspecific for BP-DNA, recognizing highly modified DNA more efficientlythan DNA modified to a low extent, may be common to all antiseraelicited against highly modified DNA immunogens. Therefore weconclude that antibody affinity must be tested also with DNAsamples of low modification, obtained either in vitro or invivo.     

Production of monoclonal antibodies to guanine imidazole ring-opened aflatoxin B1DNA, the persistent DNA adduct in vivo

Monoclonal antibodies were produced following immunisation ofmice with guanine imidazole ring-opened aflatoxin B₁ DNA (iroAFB₁ DNA), coupled electrostatically to methylated keyhole limpethaemocyanin. Three monoclonal hybridoma lines producing antibodiesspecific for iro AFB₁ DNA were grown as ascites tumours andsuitable dilutions of the ascitic fluid (1:8000–1:50 000)used in a competitive enzyme linked immunosorbent assay (ELISA)to measure reactivity of the antibodies to a variety of aflatoxinand nucleic acid-related compounds. These antibodies recogniseAFB₁ bound to DNA at levels 10⁴–10⁵ times lower concentrationthan unmodified calf thymus DNA or 8,9-dihydro-8,9-dihydroxy-aflatoxinB₁; and show 2–5 times the affinity to iro AFB₁ DNA comparedto AFB₁ DNA. The concentration of AFB₁ in iro AFB₁ DNA producing50% inhibition in a competitive ELISA was 1.8 x 10^–7 molar.Using the most sensitive hybridoma line, levels of 1 adductin 300 000 nudeotides would be detectable, which is the levelof binding found in the rat and hamster in vivo. These monoclonalantibodies should therefore prove useful in detecting theselesions in animal and human tissue samples exposed to aflatoxins.     

Quantitation of carcinogen-induced DNA damage and repair in human cells with the UVR ABC excision nuclease from Escherichia coli

Recent studies by others have shown that the endonuclease complexcoded for by the uvrA, uvrB and uvrC genes of Escherichia coli(UVR ABC excision nuclease) can incise DNA containing a varietyof ‘bulky-type’ lesions, such as those resultingfrom u.v. light, (±)-7, 8ß-dihydroxy-9, 10-epoxy-7,8, 9, 10-tetrahydrobenzo[a]pyrene (anti-BPDE), and N-acetoxy-2-acetylaminofluorene.Using partially purified UVR ABC excision nuclease, we havequantitated the number of endonuclease sensitive sites (ESS)in purified DNA isolated from human fibroblasts treated withu.v. light or BPDE. The number of ESS/10⁸ daltons of DNA werecalculated from the number average mol. wt. of the DNA as determinedby sedimentation in alkaline sucrose gradients. The number ofendonuclease sites increased linearly with increasing dosesof either u.v. light or BPDE. The UVR ABC excision nucleasewas able to incise a majority of the BPDE-DNA adducts. Xerodermapigmentosum fibroblasts, complementation group A (XP12BE) had20–25% more ESS at each dose than the BPDE-treated normal(HSBP) cells. Cells treated with 4 µM BPDE and allowed12 h of incubation to perform excision repair showed removalof 60% of the initial number of ESS from HSBP DNA and 40% ofthe ESS from XP-A DNA. Beyond 12 h XP12BE cells lost no additionalESS while HSBP cells continued to lose ESS, athough at a slowerrate, until at 48 h only 22% of the initial ESS remained. Incells treated with 10 J/m² of u.v. light, the UVR ABC excisionnuclease detected 60% of the sites recognized by the pyridiminedimer specific Micrococcus luteus glycosylase/apyrimidinic endonuclease.These results demonstrate the potential use of the UVR ABC excisionnuclease in a quantitative assay for determining the numberof carcinogen-induced lesions in human DNA.     

Induction of oxygen-dependent lethal damage by monochromatic UVB (313 nm) radiation: strand breakage, repair and cell death

The action of 313 nm radiation in cellular inactivation (biologicalmeasurements) and induction and repair of DNA strand breaks(physical measurements) were studied in a repair proficientstrain and three repair deficient strains (polA, recA, uvrA)of Escherichia coli K-12. Although the induction of breaks waslinear in purified T₄ DNA (6.3 x 10^–4 breaks/2.5 x 10⁹daltons/Jm^–2) and the polA strain (4 x 10^–4 breaks/2.5x 10⁹ daltons/Jm^–2), simultaneous induction and repairof breaks were observed in the uvrA, recA and repair proficientstrains at doses <5 x 10⁴ Jm^–2. The final rates ofinduction in these strains were 1 x 10^–4, 7.5 x 10^–5and 7.5 x 10^–5 breaks/2.5 x 10⁹ daltons/Jm^–2, respectively.A highly efficient polA-dependent repair occurring at 0°Cin minimal buffer and a second slower type of repair occurringat 31°C in the polA strain were detected. Oxygen dependenceof cellular inactivation was observed for the polA and repairproficient strains irradiated at 313 nm thus providing biologicalevidence for an oxygen-dependent lesion involved in lethalityin the short wavelength range of the solar u.v. The lower hypoxicbreak induction rates of the pol4 (1.6 x 10^–4 breaks/2.5x 10⁹ daltons/Jm^–2) and the repair proficient (3.6 x 10^–5breaks/2.5 x 10⁹ daltons/Jm^–2) strains, indicate oxygen-enhancedDNA breakage by 313 nm radiation.     

Inactivation of O6-alkylguanine-DNA alkyltransferase in the rat liver due to an intraperitoneal administration of methylated DNA

Intraperitoneal administration of 6.5 mg of in vitro methylatedDNA (meDNA) containing 1.5 x 10^–44 mM of O⁶-methylguanine(6MG) to male outbred rats weighing 150 g led to a considerabledecrease in the activity of liver O⁶-alkylguanine - DNA alkyltransferase(AT). One hour after treatment there occurred a 4- to 5-folddecrease in the AT activity followed by its slow recovery. However,after 48 h, AT activity considerably exceeded control levels.A 5-fold decrease in the amount of administered meDNA resultedin the absence of its effect, whereas administration of higheramounts produced a further AT inactivation. A similar treatmentwith non-methylated DNA did not change AT activity. The possibilityof AT exhaustion under in vivo conditions and thereby inhibitionof repair of O⁶-alkylguanine in DNA, playing a key role in mutagenic,carcinogenic and antineoplastic effects of certain alkylatingagents, might be helpful in increasing susceptibility of animalsto such compounds.     

Carcinogen-mediated induction of SV40 DNA synthesis in SV40 transformed Chinese hamster embryo cells

Exposure of SV40-transformed Chinese hamster embryo cells (lineCO50) to a series of physical and chemical carcinogens (includingactivation-dependent and activation-independent varieties) resultedin the induction of viral DNA synthesis. The carcinogen mediatedamplification of SV40 DNA was demonstrated by a highly sensitivein situ hybridization procedure for the detection of cells synthesizingSV40 DNA. Treatment of CO50 cells with an inhibitor of polycyclichydrocarbon metabolism (7,8-benzoflavone) prior to the applicationof benzo[a]pyrene or 7,12-dimethyl-benz[a]anthracene preventedthe induction of SV40 DNA synthesis, indicating that the inductiondepends upon the metabolic activation of these compounds. Non-carcinogenichydrocarbons were inactive under this assay. Two different protocolsfor determining the inducing potential of a compound are presented.The properties of this test and its possible use as a short-termassay for potential carcinogens is discussed. The possibilitythat the induction of SV40 DNA synthesis is a reflection ofa general gene amplification phenomenon mediated by carcinogensis discussed.     

Chemical carcinogen-mediated decreases in DNA 5-methylcytosine content of BALB/3T3 cells

The genomic level of DNA cytosine methylation was significantlydiminished in dividing BALB/3T3 A31 CL1–13 cells treatedwith several aromatic hydrocarbon carcinogens. Benzo[a]pyrene-induceddecreases in DNA 5-methylcytosine levels were concentrationdependent over the range of 0.1 to 1.0 µg/ml when determinedat the end of the 16 h treatment period. The enzymatic methylationof DNA cytosine residues was the most sensitive to inhibition48 h after treatment as concentrations of benzo[a]pyrene aslow as 0.033 µg/ml initiated significant reductions in5-methylcytosine levels. This inhibition of DNA cytosine methylationmay be mediated by alkylation of the DNA. Treatment of hemimethylatedDNAs with (±)-r-7, t-8-dihydroxy-t-9, 10-epoxy-7, 8,9,10-tetrahydro benzo[a]pyrene (anti-BPDE), (±)-r, 7-t-8-dihydroxy-c-9,10-epoxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene (syn-BPDE), and(±)-benzo[a]pyrene-4, 5-epoxide inhibited the transferof methyl moieties from [³H]S-adenosylmethionine to cytosineresidues in the undermethylated strand in the presence of mousespleen methyltransferase activity. The syn isomer of BPDE wasthe most potent in this action while the parent compound, benzo[a]pyrene,did not significantly decrease the methyl accepting abilitiesof treated DNAs. All chemical carcinogens that were tested andare known to transform BALB/3T3 cells initiated significantreductions in 5-methylcytosine formation by 48 h post-treatment.However, concentrations of some hydrocarbons which do not transformthese cells 5- to 50-fold above effective concentrations ofthe transforming carcinogens also provided significant reductionsin DNA cytosine methylation. Thus the inhibition of DNA methylationmay be an important step in the initiation of oncogenic transformationof BALB/3T3 cells, but decreases in DNA 5-methylcytosine levelsalone cannot account for the onset of this multi-step process.