Repair of alkylated purines in the hepatic DNA of mitochondria and nuclei in the rat

Further evidence for the preferential interaction of carcinogenswith mitochondrial DNA (mtDNA) has been obtained. In rats treatedwith high doses of N-nitrosodimethylamine (NDMA) or N-nitroso-N-butylurea(NBU), hepatic mtDNA contains 1.4 times more O⁶-methyl-2'-deoxyguanosine(O⁶-MedG) or 2.3 times more O⁶-butyl-2'-deoxyguanosine (O⁶-BudG)than does nuclear DNA (nDNA). The kinetics of removal of O⁶-MeGfrom mtDNA and nDNA are similar at both high (20 mg/kg) andlow (2 mg/kg) doses of NDMA, and the removal of O⁶-MeG can beincreased by pretreating the animals with 2-acetylaminofluorene(AAF), indicating that O⁶-MeG is repaired in the mitochondrionby a mechanism similar to that which functions in the nucleus.In contrast, O⁶-BudG is removed very slowly from mtDNA and rapidlyfrom nDNA, an observation which is consistent with the absenceof a nucleotide excision mechanism in the mitochondrion andthe repair of (O⁶-BudG, predominantly by an excision mechanism,in the nucleus of mammalian cells. A 23-kd methyltransferase(MT) protein, similar to the one found in the nucleus, has beenisolated from hepatic mitochondria and is present in mitochondriafrom which the outer membrane has been removed. It is suggestedthat O⁶-MeG, but not O⁶-BuG can be repaired from mtDNA by aMT protein that is nuclear encoded and transported across themitochondrial membrane.     

Formation and persistence of O6-ethylguanine in genomic and transgene DNA in liver and brain of {lambda}lacZ transgenic mice treated with N-ethyl-N-nitrosourea

LacZ transgenic mice are suitable for short-term muta-genicitystudies in vivo. Mutagenicity in these mice is determined inthe lacZ transgene. Since the lacZ gene is of bacterial originthe question has been raised whether DNA-adduct formation andrepair in the transgene are comparable to those in total genomicDNA. Mice were treated with N-ethyl-N-nitrosourea (ENU) andkilled at several time points following treatment. Some micewere pretreated with O⁶-benzylguanine to inactivate the repairprotein O⁶-alkylguanine-DNA alkyltransferase (AGT). O⁶-ethylguanine(O⁶-EtG) was determined in lacZ in liver and brain by meansof a monoclonal antibody-based immuno-affinity assay. In addition,O⁶-EtG and N7-ethylguanine (N7-EtG) were assayed in total genomicDNA of liver and brain with an immunoslotblot procedure. Inliver, the initial O⁶-EtG level in total genomic DNA was 1.6times that in lacZ. The extent of repair of O⁶-EtG during thefirst 1.5 h after treatment was 2.1 times that in lacZ. At latertime points, O⁶-EtG repair was the same. N7-EtG repair in genomicDNA was evident. In contrast to the liver, little repair ofO⁶-EtG in total genomic and lacZ DNA occurred in the brain whileN7-EtG was repaired. No initial difference in O⁶-EtG levelswere found in lacZ and genomic brain DNA. These findings indicatethat in the liver, total genomic DNA is more accessible thanlacZ to ENU and/or the AGT protein, during the first 1.5 h followingtreatment. Because the difference in O⁶-EtG levels in the transgeneand genomic DNA in the liver is restricted to the first 1.5h after treatment, while the fixation of mutations occurs atlater time points, O⁶-EtG-induced mutagenesis most likely isalso very similar in both types of DNA.     

The development, validation and application of a 32P-postlabelling assay to quantify O6-methylguanine in human DNA

In this study, a combined immunoaffinity purification/³²P-postlabellingprocedure has been used to quantify O⁶-methyldeoxyguanosine-3'-monophosphate(O⁶-MedGp) in human DNA. DNA digests are subjected to a two-stageimmunopurification in which the acetone-eluted fraction fromthe first stage is reapplied to a second immuno-column, andthe O6-MedGp specifically eluted using O⁶-methylguanosine (O⁶-MerG).O⁶-MedGp is then ³²P-post-labelled in the presence of deoxyinosine-3'-monophosphate(dIp) as internal standard, separated by two-dimensional TLCand levels of the adduct quantified using storage phosphor technology.The recovery of O⁶-MedGp at levels between 0.4 and 500 fmolwas 61%. Analysis of human DNA samples indicated that <1fmol O⁶-methyldeoxy-guanosine-5'-monophosphate (O⁶-MepdG) couldbe detected with a high degree of precision (coefficient ofvariation <12%) during a 2 h exposure to a storage phosphorscreen. The assay was then applied to 25 human samples fromthree separate populations, one of which was exposed to methylatingagent chemotherapy, for which O⁶-methyl-deoxyguanosine (O⁶-MedG)levels had already been quantified by HPLC/radioimmunoassay.The results indicated a high degree of correlation between thetwo assays (r = 0.99). O⁶-MedGp was detected in all the samplesanalysed with levels ranging from 0.026 to 23.2 µmol O⁶-MedGp/mol dG. The minimum amount of O⁶-MepdG detected was 0.2 fmol.As there was no detectable signal in the area to which O⁶-MepdGmaps in negative control samples, a detection limit based uponthe signal/noise ratio was impossible to quantify. However thelimit of detection of the storage phosphor technology itselfwas estimated by quantifying a visually identifiable compound,which mapped to the same region. The amount of this compoundwas determined to be 32 ± 27 amol (n = 5). If a similaramount of O6-MepdG was detected from 50 (µg of DNA, andassuming that the labelling efficiency and recovery was similarto that found in this study, then this would correspond to anadduct level of -3 nmol O⁶-MedGp/mol dG.     

Comparison of mutation spectra induced by N-ethyl-N-nitrosourea in the hprt gene of Mer+ and Mer- diploid human fibroblasts

N-Ethyl-N-nitrosourea (ENU) forms several major adducts uponreaction with DNA, of which ethylation at the O⁶ position ofguanine and the O⁴, O² and N³ positions of thymine have beenimplicated to be mutagenic lesions. To investigate what specifickinds of ENU-induced mutations were affected by the repair abilityof O⁶-alkylguanine-DNA alkyltransferase (AGT), we examined themutations in the hypoxanthlne (guanine) phosphoribosyltransferasegene (hprt) in 87 independent mutants derived from ENU-treatedAGT proficient (Mer⁺) or deficient (Men^–) diploid humanfibroblasts. Of the characterized mutations, 97% were singlebase substitutions. The major difference in the mutation spectrawas that the frequency of G·C to A·T transitionswas significantly higher in Mer^– mutants (16/38) thanin Mer mutants (4/33). The results indicate that AGT removesO⁶-ethylguanine, thus protecting human cells from parts of thecytotoxic and mutagenic effects of ENU. A high frequency ofT· to A·T transversions induced by ENU was observedin both Mer⁺ (52%) and Mer^– (34%) mutants. This type ofmutation was less frequently observed (10%) in N-methyl N'-nitro-N-nitrosoguanidine(MNNG)-induced mutants derived from the same Mer⁺ cells in ourprevious report (J. Mol. Biol., 221, 421, 1991). Comparisonof alkylating lesions formed by MNNG and ENU indicates thatO² and N³-ethylthymine are potent mutational adducts for T toA transversions. The occurrence of ENU induced T·A basepair transverslons showed a strong strand bias; 35/37 were locatedon the non-transcribed strand, assuming thymine is the mutageniclesion. The result suggests a difference in repair capacityof ethyithymine on the two strands. In addition, this type ofmutation preferentially occurred at 5'-Pu-T sequences.     

SHORT COMMUNICATION: O6-methyltransferase-deficient and -proficient CHO cells differ in their responses to ethyl-and methyl-nitrosourea-induced DNA alkylation

The mutagenic and cytotoxic effects of N-ethyl-N-nitrosourea(ENU) and N-methyl-N-nitrosourea (MNU) were compared in twoisogenic Chinese hamster ovary (CHO) cell lines differing forthe expression of the repair function for O⁶-methylguanine (O⁶-meGua),the O⁶-methyl-DNA-methyltransferase(MT). Survival and ouabainresistance (oua¹) mutation frequency were similar in the twocell lines after treatment with ENU while both effects werestrongly reduced in the MT-proficient (MT⁺) CHO cells afterexposure to MNU. The slow repair kinetics of O⁶-ethylguanine(O⁶-etGua) when compared to O⁶-meGua, i.e. 25% versus 88% removalat 20 h after treatment, could still account for the similarmutational curves reported in the two cell lines after ENU treatment.The number of ENU-induced sister chromatid exchanges (SCE) wasslightly reduced in the MT⁺as compared to MT-deficient CHO cellssuggesting a role for O⁶-etGua in SCE formation. Comparisionof survival after exposure to ENU and MNU showed that, at similarlevels of O⁶-alkylguanine on DNA, the ethyl-is more toleratedthan the methyl-adduct. These data focus the attention on theimportance of DNA damage processing in the cytotoxic responseto alkylating agents.     

Absence of {gamma}-glutamyl transpeptidase activity in neoplastic lesions induced in the liver of male F-344 rats by di-(2-ethylhexyl)phthalate, a peroxisome proliferator

Male F-344 rats were fed a diet containing 2% di-(2-ethylhexyl)phthalate(DEHP) for 95 weeks. Liver nodules and/or hepatocellular carcinomas(HCC) developed in 6/10 rats fed DEHP and none were found incontrols (P<0.005 by x² test). All the nodules and HCC werenegative for -glutamyl transpeptidase. In the non-tumorous portionsof liver, the hepatocytes contained an increased number of peroxisomesand extensive accumulation of lipofuscin. By immunocyto-chemicalanalysis, the liver peroxisomes in rats treated chronicallywith DEHP had visually detectable decrease in the H₂O₂-degradingcatalase and increase in H₂O₂-producing fatty acyl-CoA oxidase.These results show that higher dietary level of DEHP, whichcauses substantially greater degree of peroxisome proliferationthan the 1.2% dietary level used in the National ToxicologyProgram bioassay (1982, Publication no. NTP-80-37, Tech. ReportSeries No. 217), can induce liver tumors in male rats.     

Repair of O6-methylguanine, O6-ethylguanine, O6-isopropylguanine, and O4-methylthymine in synthetic oligodeoxynucleotides by Escherichia coli ada gene O6-alkylguanine-DNA-alkyltransferase

Self-complementary oligodeoxynucleotides have been synthesizedcontaining O⁶-methylguanine (O⁶meG), O⁶-ethylguanine (O⁶etG),O⁶-isopropylguanine (O⁶iprG) and O⁴-methylthymine (O⁴meT). Theyanneal in solution to give double-stranded DNA. These doublehelices have been used as substrates for the DNA repair proteinO⁶-alkyl-guanine-DNA-alkyltransferase coded for by the ada geneof Escherichia coli. The repair followed second-order chemicalkinetics. O⁶meG was repaired by the 19-kd transferase at a rateof 2.54x10⁷ M^–1 s^–1 which is close to the theoreticallimit for a diffusion-controlled reaction; O⁶etG and O⁴meT arerepaired 1000 and 10000 times more slowly. The 39-kd alkyltransferase(which is precursor to the 19-kd form) and the 19-kd transferaserepaired O⁶etG at similar rates. O⁶iprG was not repaired. Therepair of oligomers containing O⁶meG was only slightly inhibitedby the presence of non-alkylated oligomers. Oligomers containingO⁶etG were only slightly more effective as inhibitors of repairthan the non-alkylated oligomers, indicating that the transferasedoes not bind selectively to alkylated DNA. Parallel structuralstudies have shown that O⁶-alkylguanine:C and O⁴-alkylthymine:Abase pairs have a similar geometry with the alkylated base displacedinto the major groove of the DNA in contrast to O⁶-alkylguanine:Tand O⁴-alkylthymine:G base pairs which retain the Watson-Crickalignment with N1 of the purine juxtaposed to N3 of the pyrimidine.Measurement of the rate of repair of these different base pairssuggests that pairs with the alkyl group exposed in the majorgroove may be repaired more rapidly than those with the alkylgroup more deeply buried in the helix.     

Synthesis of the prototype DNA--protein cross-link, 1-(guan-1-yl)-2-(cystein-S-yl)ethane, and its role in the reactions of the haloethylnitrosoureas

The haloethylnitrosoureas form a cytotoxic DNA cross-link ina series of reactions which involves initial alkylation of theO⁶ position of guanine and rearrangement to the intermediate,1,O⁶-ethanoguanine; 1,O⁶-ethanoguanine then reacts with a neighboringcytosine base. O⁶-Alkylguanine-DNA alkyltransferase can interruptthis process after the initial alkylation step by removing thealkyl group from the O⁶ position of guanine. Recent evidencesuggests that the O⁶-alkylguanine-DNA alkyltransferase alsorecognizes 1,O⁶-ethanoguanine as a substrate, becoming boundto DNA when it interacts with that intermediate. It has alsobeen shown that glutathione becomes bound to haloethylnitrosourea-treatedDNA, apparently through chemical interaction with 1,O⁶-ethanoguanine.Since both of these reactions involve the thiol group of cysteine,we have examined the reaction of cysteine with 1,O⁶-ethanoguanine,characterizing the prototype DNA-protein cross-link, 1-(3-cytosinyl),2-(1-guanyl)ethane, which is formed in this reaction. Theseresults establish a competitive reaction with 1,O⁶-ethanoguanineas a likely route to protein-DNA cross-linking.     

O6-Ethylguanine and O6-benzylguanine incorporated site-specifically in codon 12 of the rat H-ras gene induce semi-targeted as well as targeted mutations in Rat4 cells

To examine the miscoding properties of modified guanine residuesbearing increasingly bulky O⁶-substituents, Rat4 cells, grownin the presence of O⁶-benzylguanine to deplete the DNA repairprotein O⁶-alkylguanine-DNA alkyltrans-ferase, were transfectedwith plasmids carrying H-ras genes in which O⁶-methyl, O⁶-ethyl-and O⁶-benzylguanine were substituted for the first, secondor both the first and second guanine residues of codon 12 (GGA).DNA from isolated transformed colonies was amplified by PCRand directly sequenced by high-temperature manual and automatedmethods. The results show that O⁶-ethylguanine and O⁶-benzylguanineinduced semi-targeted as well as targeted mutations, in contrastto O⁶-methylguanine, which induced only targeted mutations.When incorporated in place of the first guanine of H-ras codon12, the targeted mutations induced by all these modified guanineswere exclusively G     

A method for selection of forward mutations in supF gene carried by shuttle-vector plasmids

The supF gene of Escherichia coli has been widely used as amutagenic target in several shuttle-vector plasmids. Mutationsin this gene are usually screened by a colony colour assay basedon the suppression of a lacZ amber mutation in an appropriatebacterial indicator strain. This screening method cannot measurethe low mutation frequencies usually detected in prokaryotes,and therefore precludes the use of supF gene for studying mutationalspectra in bacteria. In this paper we report the developmentof a simple method for the selection of supF forward mutationsin shuttle-vector plasmids. The method has implied the constructionof an araD^– araC(Am) mutant strain (MBL50) of E.coli.The L-arabinose sensitivity caused by the accumulation of atoxic intermediate in araD^– mutants is abolished in MBL50because the araC(Am) mutation blocks the L-arabinose catabolicpathway. Strain MBL50 becomes sensitive to L-arabinose whentransformed with a supF⁺ plasmid but remains resistant upontransformation with a supF^– mutant. This new L-arabinoseresistance selection method was able to detect supF^– mutantfractions up to three orders of magnitude below those determinedwith the colony colour screening assay. The method was furthervalidated by carrying out in vivo mutagenesis experiments withN-methyl-N-nitrosourea (MNU) and a shuttle-vector-bearing strain(UC2109) completely defective in O⁶-methylguanine (O⁶meG) alkyltransferaserepair capacity. The DNA sequence alterations of 22 independentsupF^– mutants induced by MNU were determined. All mutationswere G:C     

Formation and persistence of benzo[a]pyrene-DNA adducts in mouse epidermis in vivo: importance of adduct conformation

The formation and repair of benzo[a]pyrene diol epoxide-N²-deoxyguanosineadducts (BPDE-N²-dG) in DNA isolated from the skin of mice treatedtopically with benzo[a]pyrene (BP) was studied by ³²P-postlabelingand by low-temperature fluorescence spectroscopy under low resolutionand under high resolution fluorescence line narrowing (FLN)conditions. In agreement with earlier studies, total BP-DNAbinding reached a maximum at 24 h after treatment (dose: 1 µmol/mouse),then declined rapidly until 4 days after treatment and muchmore slowly thereafter. An HPLC method was developed which resolvedthe ³²P-postlabeled (–)-trans- from (–)-cis-anti-BPDE-N²-dG,and (+)-trans- from (+)-cis-anti-BPDE-N² High performance liquidchromatography analysis of the major TLC adduct spot (containing>80% of the total adducts) obtained by postlabeling BP-modifiedmouse skin DNA showed that it consisted of a major componentthat coeluted with (–)-cis-/(+)-trans-anti-BPDE-N²-dGand a minor component that coeluted with (–)-trans-/(+)-cis-anti-BPDE-N²-dGand that the minor component was repaired at a slower rate thanthe major component. Low-temperature fluorescence spectroscopyof the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N²-dGand the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N²In agreement with the ³²P-postlabeling results it was observedby fluorescence spectroscopy that the (+)-cis-adducts were repairedmore slowly than most other adducts. Moreover, the (+)-trans-adductsexhibited a broad distribution of base-stacked, partially base-stackedand helix-external conformations. Mouse skin DNA samples obtainedat early timepoints (2–8 h) after treatment with BP containedsubstantially more of the ‘external’ adducts, whilesamples at later timepoints (24–48 h) contained relativelymore adducts in the base-stacked conformation, indicating alsothat the latter adducts are repaired less readily than the former.The possible biological significance of these novel observationsof conformation-dependent rates of DNA adduct repair and theirpossible dependence on DNA sequence, are discussed.     

Evidence for the excision repair of O6-n-butyldeoxyguanosine in human cells

The persistence of O⁶-n-butyldeoxyguanosine (O⁶-nBudG) in DNA,the presence of O⁶-alkylguanine DNA alkyltransferase (AT) activityin cell extracts, and cell survival following exposure to N-n-butyl-N-nitrosourea(BNU), have been measured in normal and xeroderma pigmentosumcell strains, both transformed and untransformed. The ratesof removal of O⁶-nBudG did not correlate with AT activity butdid correlate with the ability of strains to excise bulky DNAlesions. BNU and N-methyl-N-nitrosourea dose—responsecurves for cell killing suggests that both AT and excision maybe involved in the repair of cytotoxic lesions.     

Synthesis and characterization of covalent adducts derived from the binding of benzo[a]pyrene diol epoxide to a -GGG- sequence in a deoxyoligonucleotide

Direct synthesis and purification procedures are described forthe preparation of adducts derived from the covalent bindingof 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydro-benzo[a]pyrene[(+)-ante-BPDE or (+)-BPDE 2] to each of the three guanine residues(trans-N² lesions) in the oligodeoxyribonucleotide d(CTATG¹G²G³TATC)The positions of the modified Gs are defined by Maxam-Gilbertsequencing techniques. Six different oligonucleotides with oneor two precisely positioned (+)-anti-BPDE residues are identified.The absorbance, circular dichroism and fluorescence characteristicsare changed upon formation of duplexes with the complementarystrands d(GATACCCATAG). In the doubly-modified oligonucleotides,a broad, excimer-like long wavelength fluorescence emissionband is observed with a maximum near 455 nm only if the two(+)-anti-BPDE-modified Gs are adjacent to one another. The covalentlyattached (+)-anti-BPDE residues decrease the thermodynamic stabilitiesof the duplexes; their melting points are markedly dependenton the position of the lesions, being highest with the (+)-anti-BPDEresidue at G¹ (T^m=40°C, only 2°C lower than in the caseof the unmodified oligonucleotide) and lowest when it is situatedat G³ (T^m=29°C). The implications of these and other physicalcharacteristics are discussed. The facile synthesis of theseor similar site-specific and stereochemically defined (+)-trans-anti-BPDE-N²-dGlesions in runs of contiguous guanines in oligodeoxyribonucleotidesof specified base sequence should be useful for the design ofsite-directed mutagenesis studies in vitro and in vivo.     

The role of O6-alkylguanine-DNA alkyltransferase in protecting Rat4 cells against the mutagenic effects of O6-substituted guanine residues incorporated in codon 12 of the H-ras gene

The role of rat O⁶-alkylguanine-DNA alkyltransferase (AGT) inmodulating mutagenesis by O⁶-substituted guanines in the ratH-ras gene was examined. Rat4 cells were transfected with vectorscarrying O⁶-methyl-, O⁶-ethyl- or O⁶-benzylguanine residuesin place of the normal guanines at either the first, second,or both the first and second positions in codon 12 (GGA) ofthe H-ras coding sequence. The percentage of transformed colonieswas determined for cells grown in normal medium or in mediumcontaining O⁶-benzylguanine to completely deplete AGT. In parallelexperiments with O⁶-methylguanine-containing vectors, the percentageof cellular DNA harboring codon 12 mutations was determinedfor normal cells and cells lacking AGT. A reasonable correspondencewas observed between the percentage of mutated DNA and the percentageof transformed colonies produced in both types of cells. Theresults indicate that the contribution of AGT to the repairof O⁶-substituted guanine damage decreases as the O⁶ substituentis changed from methyl > ethyl > benzyl. Additionally,cellular AGT appears to repair an O⁶-methylguanine more readilyat the first position of codon 12 than the second position.However, other repair mechanisms in these mammalian cells appearto play a major role in correcting low levels of O⁶-substitutedguanine damage including O⁶-methylguanine damage.     

Alkylation of DNA in F344 rat thymus following administration of 1-n-propyl-1-nitrosourea in vivo and comparison of O6-alkylguanine DNA alkyltransferase activity in thymus from F344 and Long-Evans rats in vitro

The tissue distribution of radioactivity 1 h after i.p. injectionof [n-propyl-2,3-³H]1-n-propyl-1-nitrosourea (PNU) (100 mg/kg)was studied in male F344 rats. This treatment results in a highincidence of thymic lymphomas. The ³H concentration in the thymus,testis and brain was significantly higher than that in blood.7-n-propylguanine and O⁶-n-propylguanine were detected in thymusDNA of F344 rats treated with PNU; the ratio of O⁶-n-propylguanine/7-n-propylguaninewas 0.35, lower than following DNA alkylation in vitro. Thissuggests the presence of O⁶-alkylguanine DNA alkyltransferase(AGT) in thymus. AGT activity in F344 and Long-Evans rats wascompared by using a ³H-propylated DNA as a substrate. AGT activityin the thymus of F344 rats was lower than that in the liver.The AGT activity in the thymus of Long-Evans strain, which hada low incidence of PNU-induced thymic lymphomas, was higherthan that of F344 strain. The high level of DNA alkylation byPNU and the low activity of AGT in the thymus may contributeto the high incidence of thymic lymphoma in F344 rat.     

Formation and persistence of O6-(2-hydroxyethyl)-2'-deoxyguanosine in DNA of various rat tissues following a single dose of N-nitroso-N-(2-hydroxyethyl)urea. An immuno-slot-blot study

Rabbit antibodies against O⁶-(2-hydroxyethyl)-2'-deoxyguanosine(O⁶-HEdG) were used to develop a highly sensitive immuno-slot-blotassay for this promutagenic base which enabled the quantitationof 3.6 µmol O⁶-HEdG/mol deoxy-guanosine, correspondingto 5 fmol in a 3-µg DNA sample. This assay was used tostudy DNA hydroxyethylation by N-nitroso-N-(2-hydroxyethyl)urea(HENU) in adult male F344 rats. Initial amounts of O⁶-HEdG 2h after a single i.v. dose of 50 mg/kg were highest in kidney(81 µmol O⁶HEdG/mol deoxyguanosine), followed by lungand liver (67 and 55 µmol/mol dG respectively). Formationof O⁶-HEdG in cerebral DNA was considerably lower (18 µmolO⁶-HEdG/mol deoxyguanosine), probably reflecting delayed crossingof the blood—brain barrier by HENU due to its hydrophilicity.The formation of O⁶-HEdG in liver and kidney was strictly proportionalto dose over a range of 5–50 mg HENU/kg. Repair of O⁶-HEdGwas very rapid in liver (apparent half-life, 12 h), and somewhatslower in kidney and lung (approximate half-life, 40 h and 48h respectively). In contrast, 62% of the initial amount of O⁶-HEdGin cerebral DNA was still present after 7 days. Saturation ofthe hepatic O⁶-alkyl-guanine-DNA alkyltransferase by pretreatmentwith N-nitrosodimethylamine (20 mg/kg) almost completely inhibitedthe removal of O⁶-HEdG, indicating that O⁶-HEdG is predominantlyrepaired by this repair enzyme.     

Cell specific differences in O6-methylguanine-DNA methyltransferase activity and removal of O6-methylguanine in rat pulmonary cells

Previous studies have demonstrated that cell specificity existsfor the alkylation of DNA from lung cells following treatmentof rats with the tobacco specific carcinogen 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone(NNK). The concentration of the promutagenic adduct O⁶-methylguanine(O⁶MG) was found to be greatest in Clara cells followed by macrophages,type II cells and alveolar small cells. The purpose of thisstudy was to measure the activity of the repair protein O⁶-methylguanine-DNAmethyltransferase (O⁶MGMT) and to determine whether differencesexist for the removal of O⁶MG among pulmonary cell types. Constitutiveactivity of O⁶MGMT was 2-fold greater in macrophages and typeII cells than alveolar small cells and Clara cells. Treatmentfor 4 days with NNK (10 mg/kg/day) had no effect on O⁶MGMT activityin macrophages, but decreased activity in alveolar small cellsand type II cells by 57 and 84%, respectively. O⁶MGMT activitywas reduced to below limits of detection in Clara cells followingtreatment with NNK. The effect of NNK on O⁶MGMT activity wasconsistent with rates of removal of O⁶MG in macrophages andClara cells. The loss of O⁶MG from DNA of macrophages followedfirst order kinetics (t = 48 h) while very little loss of thisadduct was observed in Clara cells over an 8 day period followingcessation of carcinogen treatment. Even though O⁶MGMT activitywas reduced in alveolar small cells and type II cells, 90% ofthe O⁶MG bound to DNA in these cell types was removed within8 days after treatment was discontinued. The loss of O⁶MG frompulmonary cells appears to result largely from the removal ofthis adduct by O⁶MGMT since rates of cell turnover were verylow (0.5–1.5%/day) in the lung and were not affected bytreatment with NNK. This study indicates that the activity ofO⁶MGMT and the rate of resynthesis of this repair enzyme differconsiderably among pulmonary cells following the methylationof DNA. The high concentration of O⁶MG in Clara cells and thelow rate of repair of this promutagenic adduct may be criticalfactors in the potent pulmonary carcinogenicity induced by thetobacco specific carcinogen NNK.     

Inhibition of the metabolism of N-nitrosoacetoxymethylmethylamine in the rat by disulfiram

The stability of N-nitrosoacetoxymethylmethylamine (NAMM) inrat serum in vitro, the half-life in blood in vivo and the exhalationrate of ¹⁴CO₂ after the application of ¹⁴C-labelled NAMM werestudied with and without disulfiram (DSF) pre-treatment. Itwas found that the metabolism of NAMM is inhibited by DSF invitro as well as in vivo. The influence is thought to be dueto inhibition of esterase activity, as shown by comparing thekinetics of the degradation of NAMM with those of p-nitrophenylacetate.In contrast to this result the exhalation of ¹⁴CO₂ does notseem to be influenced by the esterase inhibiting effect of DSF.     

DNA adduct formation in Salmonella typhimurium, cultured liver cells and in Fischer 344 rats treated with o-tolyl phosphates and their metabolites

2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilicand a neurotoxic metabolite of o-tolyl phosphates. In a previouspaper we reported that 2-phenoxy-4H-1,3,2-benzodioxaphosphorin2-oxide is mutagenic in Salmonella typhimurium TA100 and formsDNA adducts in incubations with nucleotides, nucleosides andisolated DNA. In the present study we compare DNA adduct formationusing ³²P-post-labelling assays in 2-phenoxy-4H-1,3,2-benzodioxaphosphorin2-oxide-treated bacteria (S.typhimurium TA100) and hepatomacells with DNA adducts formed in liver, kidney, lung and heartof tri-o-tolyl phosphate-exposed Fischer 344 male rats. In bothbacteria and hepatoma cells two DNA adducts could be detectedafter treatment with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin2-oxide. The minor adduct co-chromatographed with syntheticN³-(o-hydroxy-benzyl)deoxyuridine 3' monophosphate after postlabelling.The major DNA adduct was a cytidine adduct, most likely N³-(o-hydroxybenzyl)deoxycytidine3' monophosphate. Male Fischer 344 rats were treated orallyfor 10 days with tri-o-tolyl phosphate (50 mg/kg/day) and DNAwas isolated from liver, kidney, lung, heart, brain and testes1,4,7 and 28 days after giving the last dose. Analysis by ³²P-postlabellingrevealed that two adducts were present in the DNA isolated fromliver, kidney, lung and heart on the first day after givingthe last dose; DNA adducts were not detected in the brain andtestes. The adduct pattern after in vivo treatment with tri-o-tolylphosphate was identical with that found in bacteria and hepatomacells treated with 2-phenoxy-4H-1,3,2-benzo-dioxaphosphorin2-oxide, the major adduct being N³-(o-hydroxybenzyl)deoxycytidine3' monophosphate and the minor N³-(o-hydroxybenzyl)deoxyuridine3' monophosphate. Both DNA adducts persisted in the lungs forthe entire observation period, whereas in the kidney only thecytidine adduct could be detected 28 days after the last doseof tri-o-tolyl phosphate. In liver and heart the adducts weredetectable only on the first day after completion of the treatment.The results indicate that in addition to the well establishedneurotoxicity, some o-tolyl phosphates may have a carcinogenicpotential.     

A human cell line proficient in O6-methylguanine-DNA-methyltransferase and hypersensitive to alkylating agents

The involvement of O⁶-methylguanine (O⁶-meGua) in mutagenesisis well established, while the toxic effect of these residuesis still controversial. In this study, we compare the cytotoxicityof N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea(MNU) on three cell lines of different origin, which have differentabilities to repair O⁶-meGua residues (Mer phenotype): a humanhepatoma cell line (LICH cells, Mer⁺), a rat hepatoma cell line(H4 cells, Mer⁺) and a Chinese hamster cell line (CHO cells,Mer^– phenotype). LICH and CHO cells show the same sensitivityto the killing effect of MNNG and MNU and are 5-fold more sensitivethan H4 cells. However, LICH and H4 cells share similar sensitivitiesto the toxic effect of 1, 3-bis(2-chloroethyl)-l-nitrosourea.O⁶-meGua residues are removed at the same rate from the DNAof [³H]MNU-treated LICH and H4 cells, which also do not differin the rate of removal of N3-methyladenine residues nor in overallDNA repair synthesis. The results suggest that MNNG and MNUproduce a lethal lesion that is repaired by a process that doesnot involve the alkyltransferase.