Tissue and cell specific methylation, repair and synthesis of DNA in the upper gastrointestinal tract of Wistar rats treated with single doses of N-methyl-N'-nitro-N-nitrosoguanidine

Several potential cancer risk factors have been monitored concurrentlyin the upper gastrointestinal tract of young adult male Wistarrats given single (i.g.) doses of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) which readily induces forestomach tumours under theseconditions. Radioimmunoassay was used to determine the formationof O⁶-methy1-2'-deoxyguanosine (O⁶-MedG) in DNA after MNNG dosesof 1, 5, 25 or 50 mg/kg and was found to be highest in the pylorus,with progressively lower levels in the corpus, forestomach,duodenum, oesophagus and jejunum. Immunohistochemical proceduresshowed that cells with nuclei containing O⁶-MedG were heterogeneouslydistributed in these tissues. O⁶-Alkylguanine-DNA alkyltransferaseactivity in untreated animals was highest in the mucosae ofthe corpus, lower and relatively similar in that of the pylorus,duodenum and jejunum and lowest in the tissues of oesophagusand forestomach. Estimates of DNA synthesis and cell proliferationindicated a 5-fold increase in the DNA labelling index in theforestomach whereas perturbations of DNA synthetic activityin the other tissues of the upper gastrointestinal tract weremuch less marked. As a result of these changes, cells with nucleithat contained O⁶-MedG and were also undergoing DNA synthesis(determined by sequential immunohistochemical analysis and autoradiography)were found most commonly in the forestomach and to a lesserextent in the pylorus. This distribution of replicating damagedcells corresponds with the relative tumour yields in these uppergastrointestinal tract tissues and such cells are the probabletargets in this single dose carcinogenesis regime. Thus, whilstthe highest concentration of O⁶-MedG did not correlate tumourincidence, the overall risk for tumour induction did correlatewith a significant level of DNA damage, a lower capacity forDNA repair and a marked increase in DNA synthesis over the constitutivelevel in the target cells. Carcinogenic risk in this systemis therefore more readily determined by studying several riskfactors simultaneously.     

Tissue and cell specific methylation, repair and synthesis of DNA in the upper gastrointestinal tract of Wistar rats treated with N-methyl-N'-nitro-N-nitrosoguanidine via the drinking water

Several potential cancer risk factors have been monitored concurrentlyin the upper gastrointestinal tract of young male Wistar ratsgiven N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) via the drinkingwater, a regimen that induces a high yield of tumours in thepylorus and to a lesser extent in the duodenum. Radioimmunoassaywas used to determine the amounts of O⁶-methy1-2'-deoxyguanosine(O⁶-MedG) formed in the tissue DNA of rats given MNNG at dosesof 40 or 80 µg/ml for periods of 3, 6 and 12 weeks. Thehighest adduct concentration was found in the pylorus with progressivelylower concentrations in the corpus and duodenum, jejunum, forestomachand oesophagus. Between 3 and 12 weeks these adduct levels decreasedin all tissues and there was no evidence of a dose dependentaccumulation of O⁶-MedG. When analysed by immunohistochemistrythe distribution of cells with nuclei containing O⁶-MedG wasseen to be heterogeneous in the various tissues. O⁶-Alkylguanine-DNAalkyltransferase activity increased during the 12 weeks of MNNGtreatment in oesophagus and forestomach, but decreased to 50%of the initial value in the corpus, pylorus, duodenum and jejunum.The major changes in DNA synthesis and cell proliferation werethe marked upward expansion (i.e. towards the lumen) of thezone of replicating cells in the glands of the pylorus and thegreatly increased numbers of replicating damaged cells (i.e.cells that contained O⁶-MedG whilst undergoing DNA synthesis)as determined by sequential immunohistochemical analysis andautoradiography. Such cells are the probable target cells inthis chronic dose carcinogenesis regime. Although similar changesalso occurred in the glands of the corpus these were of lesserextent and the changes of labelling index in the oesophagusand forestomach were relatively minor. In the duodenum, MNNGtreatment led to erosion of the upper part of the glands sothat the zone of cells containing O⁶-MedG overlapped with thezone of proliferating cells resulting in the formation of manyreplicating damaged cells. Thus, as in the single dose study(see preceding paper) the distribution of replicating damagedcells coincides with the tumour yield in the tissues of theupper gastrointestinal tract. As in the case of single dosesof MNNG the risk factors for carcinogenesis are, a significantlevel of DNA damage, a lower capacity for DNA repair and anincreased DNA synthetic activity, again suggesting that carcinogenicrisk cannot readily be determined by studying risk factors individually.     

DNA methylation in rat stomach and duodenum following chronic exposure to N-methyl-N'-nitro-N-nitrosoguanidine and the effect of dietary taurocholate

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces a high incidenceof carcinomas in the glandular stomach of rats following chronicadministration in the drinking water. We determined the levelof 7-methylguanine and O⁶-mehtylguanine in gastric and duodenalDNA during chronic exposure to MNNG (80 p.p.m.). After considerablefluctuations during the initial 3 weeks, levels of methylpurinesreached a steady state which was approximately three times higherin the pylorus (i.e. the preferential site of tumor induction)thanin the fundus and duodenum, with 7-methylguanine and O⁶-methylguaninevalues in the rangeof 520 and 110 µmol/mol guanine, respectively.When rats were given MNNG in the drinking water at concentrationsranging from 10 to 80 p.p.m. for 3 weeks, levels of methylpurinesreached maximum values already at 10–20 p.p.m. At higherMNNG concentrations, there was no further increase in DNA alkylation.The reason for this lack of dose response remained unclear.Immunohistochemical analyses showed that DNA methylatlon byMNNG is restricted to epithelial cells bordering the luminalsurface. The possibility exists that in this target cell populationthe content of free thiols is a limiting factor for the decompositionof MNNG and its reaction with macromolecules in the gastricmucosa. Addition to the diet of sodium taurocholate, a bileacid previously shown to enhance MNNG-induced stomach carcinogenesis,did not influence the extent of DNA methylation, indicatingthat it acts as a promoter.     

The O6-methylguanine-DNA activity of rat hepatoma cells is increased after a single exposure to alkylating agents

The O⁶-methylguanine-DNA-methyltransferase activity was measuredin rat hepatoma cells (H4 cells) at different times after N-methyl-N'-nitro-N-nitrosoguanidine(MNNG), methyl- methane sulfonate (MMS) or ethylinethane sulfonate(EMS) treatment. Incubation with MNNG (10µM) first depletesthe methyltransferase activity, then the number of methyltransferasemolecules per cell increases and reaches-3-fold the constitutivelevel after 48 h. Incubations with MMS (0.5 or 1 mM) or withEMS (5 or 10 mM) do not modify or partially decrease the constitutivemethyltransferase level. However, an enhancement of the activityis also observed after 48 h: the activity is 5-and 4-fold higherthan the control value in MMS-and EMS-treated cells, respectively.The methyltransferase increase is due to de novo protein synthesis.It is not observed in cells constitutively lacking this protein.The data suggest that the O⁶-methylguanine (O⁶-meGua) repaircapacity of H4 cells can be increased after a single treatmentwith alkylating agents, by a process different to the adaptiveresponse.     

Promutagenic methylation damage in liver DNA of mice infected with Schistosoma mansoni

Male and female BK-TO mice were infected with different numbersof Schistosoma mansoni cercariae under standard environmentalconditions. Promutagenic methylation damage (O⁶-methyldeoxyguanosine;O⁶-MedG) was detected in liver DNA, but not in kidney, spleenor bladder DNA of infected animals. It was shown that levelsof hepatic O⁶-MedG increased with increasing intensities ofschistosomal infection. Possible mechanisms of action are discussed.These include the activating effects of schistosomes and theirproducts on murine macrophages and subsequent endogenous formationof N-nitroso compounds by the activated macrophages.     

MNNG-induced partial phenotypic reversion of Mer- cells

The effect of pretreatment with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) on MINNG sensitivity of the surviving population wascompared in two HeLa lines, one of the Mer⁺ phenotype (HeLaS3) and one of the Mer^– phenotype (HeLa MR). Whereas MNNGpretreatment of HeLa Mer⁺ cells had no effect on the MNNG sensitivityof surviving cells, Mer^– cells surviving a first exposureto MNNG became much more resistant to MNNG Comparison of thesensitivity of individual HeLa MR clones with their MNNG-pretreatedpopulation and analysis of the composition of the pretreatedpopulation showed that the majority of cells surviving the MNNG-pretreatmentnow displayed the Mer⁺ phenotype in respect to sensitivity toMINNG. One MNNG-resistant clone derived from a pretreated HeLaMR population (Cl 4) was characterized further. It had a similarsensitivity to the Mer⁺ line to all monofunctional alkylatingagents, but was as sensitive as the Mer^– line to the crosslinkingagent chloroethylntrosourea. Cl 4 cells, like the Mer^–cells, did not repair O⁶-methylguanine (O6MeG). The resultssuggest that the two characteristics which are usually coupledwith the Mer^– phenotype - lack of O⁶MeG repair and hypersensitivityto MNNG - can be separated.     

Penetration of N-methyl-N'-nitro-N-nitrosoguanidine to proliferative cells in gastric mucosa of rats is different in pylorus and fundus and depends on exposure time and solvent

We have studied the penetration of a labeled gastric carcinogen,N-(³H)methyl-N'-nitro-N-nitrosoguanidine (³H-MNNG), from thegastric lumen to proliferative cells in the gastric mucosa ofWistar rats. ³H-MNNG was dissolved in deionized water or dimethylsulfoxide (DMSO) and given intragastrically through a tube inthe forestomach. Cells in the S-phase were labeled by incorporationof bromodeoxy-uridine. Penetration of the carcinogen was evaluatedby light microscopy after immunohistochemistry and autoradiography.Cells in S-phase labeled with ³H-MNNG (double-labeled cells)are considered to represent the cell population at risk of MNNG-inducedcarcinogenesis. When deionized water was used as solvent forthe carcinogen, the average percentage of double-labeled cellsin the pylorus was 12.0, 22.4 and 32.5 respectively, after 10,30 and 60 min of mucosal exposure to ³H-MNNG. The correspondingpercentages for the fundus mucosa were 1.7, 3.1 and 3.4, whichare significantly lower than the pylorus values. When DMSO wasused as solvent for the carcinogen, the percentage of double-labeledcells was 0.3 and 1.1 in the pylorus and 0.1 and 1.3 in thefundus after 10 and 30 min exposure to ³H-MNNG. Dimethyl sulfoxidecaused superficial mucosal damage to the gastric mucosa andincreased secretion of fluid into the stomach. Our results stronglysuggest that gastric cancer develops in the pylorus becausethe carcinogen penetrates more easily into pyloric than fundicmucosa. The results support the view that delayed gastric emptyingis a risk factor in gastric carcinogenesis, and show that DMSOcounteracts the penetration of MNNG into the mucosa.     

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.     

N-Methyl-N'-nitro-N-nitrosoguanidine-resistant HeLa S3 cells still have little O6-methylguanine-DNA methyltransferase activity and are hypermutable by alkylating agents

To clarify the involvement of O⁶-methylguanine (O⁶-MeG) in mutagenesis,we isolated N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-resistantcells, MR10-1 from HeLa S3 mer^– cells. MR10-1 cells were40 times more resistant to MNNG than the parental cells. MR10-1cells were also significantly more resistant to N-methyl-N-nitrosoureaand slightly more resistant to methyl methanesulfonate and dimethylsulfate than parental cells. However, we found that MR10-1 cellshad still little O⁶-MeG-DNA methyltransferase activity and weresensitive to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosoureahydrochloride, like HeLa mer^– cells, thereby showing thatMR10-1 cells are still mer^–. When induced 6-thioguanine(6TG)-resistant colonies were plotted as a function of the correspondingpercentage survival, the resistant colonies of MR10-1 cellswere induced much more frequently than in the case of HeLa mer^–cells. However, induction of 6TG-resistant cells in the bothcell lines did not differ significantly in terms of mutant cellsper 0.1 µM MNNG. On the contrary, MR10-1 cells (mer^–)and two HeLa S3 mer⁺ cells lines differed in the induction ofmutation as a function of MNNG concentration. The HeLa mer⁺cell lines were not mutable, while MR10-1 cells were highlymutable. These above results clearly show that the HeLa mer^–cell has at least two defects in the repair of the alkylatedadducts which are related to cell killing and mutation, andalso suggest that O⁶-MeG is involved in the induction of mutation.     

Transfectant CHO cells expressing O6-alkylguanine-DNA-alkyltransferase display increased resistance to DNA damage other than O6-guanine alkylation

BCNU [1,3-bis(2-chloroethyl)-1-nitrosourea, Carmustine] is anitrosourea that crosslinks DNA and is useful in cancer chemotherapy.Tumor cells resistant to BCNU produce high levels of O⁶-alkylguanine-DNA-alkltransferase(AT), a protein that removes the O⁶-guanine adduct formed byBCNU prior to crosslinking. By the transfection of a human cosmidlibrary into the Chinese hamster ovary cell line AA8, severaltransgenic cell lines which express the AT gene have been constructed.These ‘BR’ cells were isolated on the basis of theirresistance to G-418 and BCNU. Like human mer⁺ strains, BR cells(relative to the parental AA8 cells) are {small tilde}500 timesmore resistant to the cytotoxic effects of 80 µM BCNU.Treatment with exogenous O⁶-methylguanine (O⁶MG), which depletescellular AT, abolishes their BCNU resistance. Also consistentwith the mer⁺ phenotype, BR cells are resistant to the mutagenicand killing activity of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG). Treatment with exogenous O⁶MG, while reversing the resistanceto MNNG mutation, does not reverse the resistance to MNNG killing.Unexpectedly, BR cells also exhibit resistance to killing bydimethylsulfate (DMS). The BR cells are not, however, detectablyresistant to UV light. These results suggest that AT activityin mammalian cells is dosely linked to the activity of otherDNA repair pathways.     

Limited capacity for the removal of O6-methylguanine and its regeneration in a human lymphoma line

The Raji human lymphoma line is able to remove O⁶-methylguanine(O⁶MeG) lesions introduced by treatment of cells with N-methyl-N'-nitro-N-nitroso-guanidine(MNNG). The reaction has a rapid phase in which 40% of theO⁶MeG is removed in the first 10 min. The capacity of cellsfor rapid O⁶MeG removal is limited and is saturated at concentrationsof MNNG which do not saturate the systems removing 3-methyladenine.Pretreatment of cells with MNNG inhibits their ability to removeO⁶MeG produced by a subsequent dose given after 2 h. Treatmentwith N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) is effectivein diminishing cellular capacity for O⁶MeG removal, and cellsunable to remove O⁶MeG and sensitive to the cytotoxic effectsof MNNG are also more sensitive to ENNG than their removal competentcounterparts. Regeneration of the ability to remove O⁶MeG requiresincubation of cells for periods > 24 h. The O⁶MeG removalsystem is similar to that found in adapted Escherichia colialthough the capacity of the Raji lymphoma line is much lowerthan that of the induced bacteria per unit of DNA.     

Increased O6-methylguanine-DNA methyltransferase activity and reduced mutability in 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea-resistant HeLa S3 cells

To clarify the involvement of O⁶-methylguanine (O⁶-MeG) in mutagenesis,we have been trying to isolate Mer⁺ cells from a HeLa S3 Mer^–cell line, and to compare the mutation frequencies between thecell lines. We previously isolated the N-methyl-^N'-nitro-^N-nitrosoguanidine(MNNG)-resistant cells, MR10–1, from HeLa S3 Mer^–cells. However, the MR10–1 cells still had only a littleO⁶-MeG-DNA methyltransferase (MT) activity. In the present study,we have isolated two 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-mitrosourea(ACNU)-reslstant cells, AC^r41 and AC^r42, from the MR10–1cells. The two AC^r cells had increased MT activities. The AC^rcells were also significantly more resistant to 1-(2-chloroethyl)-1-nitrosoureaand slightly more resistant to MNNG than the MR10–1 cells.When the mutation frequencies were tested at the hypoxanthine–guanine phosphoribosyl transferase and ouabain loci in thesecell lines, the two AC^r cells were more resistant to the mutageniceffect of MNNG than the MIR10–1 cells. These results showthe linkage between the resistance to the cytotoxic effect ofnitrosourea compounds and MT activity, and strongly supportthe hypothesis that O⁶-MeG is the main pre-mutagenic lesioninduced by MNNG.     

Isolation of clones displaying enhanced resistance to methylating agents in O6-methylguanine-DNA methyltransferase-proficient CHO cells

O⁶-Methylguanine-DNA methyltransferase (MT)-proficient Chinesehamster ovary cells were grown in the presence of low, graduallyincreasing levels of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)with the aim of selecting MNNG resistant cell lines. Six resistantclones with two levels of resistance were isolated. A 3-foldincrease in survival was observed in clones 13, 14 and 15 anda > 10-fold increase in clones A, B and C. Cross resistanceto N-methyl-N-nitrosourea but not to mitomycin C was observed.By comparison with the parental MT-proficient cells, MT activitywas doubled in two resistant clones (13 and B) irrespectiveof then- resistance levels. DNA glycosylase activity responsiblefor the removal of 7-methylguanine and 3-methyladenine showedsimilar levels in resistant clones 13 and B, in the MT-proficientcells and in the original MT-deficient cells. Alkylation-inducedDNA damage, as measured by alkaline elution at the same MNNGdose, was higher in clones 13 and B than in the parental cells.The induction of sister chromatid exchanges by MNNG was inverselyrelated to the resistance levels, thus paralleling the inductionof cytotoxicity. These results suggest the existence in Chinesehamster ovary cells of at least two independent functions whichcontrol resistance to methylating agents, one possibly beingthe capacity to repair O⁶-methylguanine.     

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.     

O6-methylguanine induces intrachromosomal homologous recombination in human cells

N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which alkylatesmany positions in DNA including the O⁶ position of guanine,efficiently induces intrachromosomal homologous recombinationin mouse L-cells. To investigate the role of O⁶-methylguaninein the induction of homologous recombination in human cells,three cell strains containing duplicated copies of the Herpessimplex virus I thymidine kinase (Htk) gene and three cell strainscontaining duplicated copies of the gene coding for hygromycinphosphotransfer-ase (hyg) were treated with MNNG. Neither theHtk genes nor the hyg genes code for a functional enzyme becauseeach contains an insertion mutation at a unique site, i.e. 8-bpXhoI linker insertions in the Htk genes and 10-bp HindIII linkerinsertions in the hyg genes. These cell strains differ in theirlevel of O⁶-alkylguanine-DNA alkyltrans-ferase (AGT), whichspecifically removes the methyl group from the O⁶ position ofguanine. Generation of a functional Htk or hyg gene has beenshown to require intrachromo-somal homologous recombinationbetween the two mutant Htk genes or the two mutant hyg genes.In all six cell strains, MNNG induced a dose-dependent increasein the frequency of homologous recombination. In each set, therewas an inverse correlation between the frequency of MNNG-inducedrecombination and the level of AGT activity. To further studythe role of O⁶-methylguanine in the induction of homologousrecombination, we used O⁶-benzylguanine to inactivate AGT intwo additional human cell strains containing the hyg recombinationsubstrate. After depletion of AGT activity by O⁶-benzylguanine,both cell strains showed a significantly elevated level of MNNG-inducedhomologous recombination. These results indicate that O⁶-methylguanineis the principal lesion responsible for the induction of homologousrecombination in these human cells by this methylating agent.     

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.     

Cytotoxicity, mutations and SCEs induced by methylating agents are reduced in CHO cells expressing an active mammalian O6-methylguanine-DNA methyltransferase gene

Alkylation at the O⁶ position of guanine leading to miscodingduring DNA replication has been shown to correlate with mutagenesisboth in bacteria and mammalian cells. The widely used Chinesehamster ovary cells (CHO) are unable to remove O⁶-meyhylguanine(O⁶-meG) due to the absence of O⁶ DNA methyltransferase (MT)activity. Recently Ding et al. [Mol. Cell. Biol. (1985) 5, 3293–3296]transfected CHO cells with human liver DNA obtaining a lineprovided with a function for the repair of O⁶-meG. We confirmedthe presence of MT activity in this particular clone (14 300molecules/cell). We used this MT-proficient cell line as comparedwith the original MT-deficient CHO cell line to analyse therelevance of repair of this lesion on cell killing, ouabain^{resistance (oua}r) mutations and sister chromatid exchanges (SCEs)induced by methylating agents. MT-proficient cells were moreresistant than MT-deficient ones to the cytotoxic and mutageniceffects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea(MNU). Furthermore a lower number of MNNG-induced SCEs werefound in MT-proficient CHO than in MT-deficient cells. Similaroua^r mutation frequencies were recorded in the two cell linesafter 4-nitroquinoline-1-oxide (4NQO) treatment showing thatthe differences in cytotoxicity and mutagenesis are restrictedto treatment with alkylating agents.     

Effect of salt-induced mucosal damage and healing on penetration of N-methyl-N'-nitro-N-nitrosoguanidine to proliferative cells in the gastric mucosa of rats

We have studied the effect of gastric exposure to 4.5 M NaClon penetration of a carcinogen, N-[³H]methyl-N'-nitro-N-nitrosoguanidine(³H-MNNG) from the gastric lumen to proliferative cells in thegastric mucosa of Wistar rats at different time intervals aftersalt exposure. Cells in S-phase were labeled by incorporationof bromodeoxyuridine. Cells in S-phase labeled with ³H-MNNG(double-labeled cells) are the cell population at risk of N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)-induced gastric carcinogenesis. Ten minutes after saltdamage the average percentage S-phase cells labeled with ³Hin pylorus was significantly decreased compared to control (1.2± 0.6 and 9.5 ± 0.7). Ten minutes after salt exposurea marked increase in gastric mucosal blood flow and leakageof fluid from the mucosa into the gastric lumen were observed,and the damaged gastric mucosa was covered by a thick mucoidlayer. These factors may contribute to the reduced ³H penetrationinto mucosa immediately after damage. Two hours after salt exposurethe number of double-labeled cells (8.6 ± 3.7/mm) andpercentage S-phase cells labeled with ³H-MNNG (10.4 ±3.1) in pylorus did not differ from control (6.1 ± 0.9/mmand 9.5 ± 0.7). Twelve and 24 h after salt exposure thenumber of double-labeled cells (79.6 ± 13.4/mm and 32.4± 2.4/mm) and the percentage S-phase cells labeled with³H-MNNG (29.7 ± 2.8 and 18.9 ± 1.3) in pyloruswere significantly increased compared to control. Increasednumber of S-phase cells, a higher location of the proliferativezone in the glandular layer were observed 12–24 h aftersalt exposure and increased permeability of the mucosa to carcinogenwas observed 12 h after salt exposure. These factors explainthe increased number of double-labeled cells and the increasedpenetration of carcinogens to the proliferative cells, and maycontribute to explain the previously described cocarcinogeniceffect of salt on gastric carcinogenesis.     

Expression of the inactive C145A mutant human O6-alkylguanine-DNA alkyltransferase in E.coli increases cell killing and mutations by N-methyl-N'-nitro-N-nitrosoguanidine

Human O⁶-alkylguanine-DNA alkyltransferase (AGT) counteractsthe mutagenic and toxic effects of methylating agents such asN-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by removing themethyl group from O⁶-methylguanine lesions in DNA. The methylgroup is transferred to a cysteine acceptor residue in the AGTprotein, which is located at residue 145. The C145A mutant ofAGT in which this cysteine is converted to an alanine residueis therefore inactive. When this C145A mutant was expressedin an Escherichia coli strain lacking endogenous alkyltransferaseactivity, the number of G:C