The induction of SCE and chromosomal aberrations with relation to specific base methylation of DNA in Chinese hamster cells by N-methyl-N-nitrosourea and dimethyl sulphate

Chinese hamster cells (V79) were treated, either as exponentiallyproliferating cultures or under conditions where they were density-inhibited,with various doses of the potent carcinogen N-methyl-N-nitrosourea(MNU) or the relatively weak carcinogen dimethylsulphate (DMS).The colony forming ability of these cells and the induced frequenciesof sister chromatid exchanges (SCEs) and chromosomal aberrationswere assayed. Following the exposure of density-inhibited cellsto radio-labelled methylating agents (labelled in the methylgroup) these phenomena were related to the levels of 7-methylguanine(7-meGua), O⁶-methylguanine (O⁶-meGua) and 3-methyladenine (3-meAde)in the DNA. At equitoxic doses MNU and DMS induced similar frequenciesof SCEs and chromosomal aberrations. Since, at equitoxic doses,MNU produces 20 times more O⁶-meGua in V79 cell DNA than doesDMS, this indicates that the formation of O⁶meGua in DNA isnot a major cause of SCEs and chromosomal aberrations. DMS-inducedSCEs may be mediated via the production of both 3-meAde and7-meGua in the DNA; these two methylated purines may also beresponsible for MNU-induced SCEs. Therefore, no one specificmethylated purine was identified as being solely accountablefor the formation of SCEs. Also, the repair of lesions in theDNA of non-replicating V79 cells leads to a reduction in theSCE frequency on their subsequent release from the density-inhibitedstate, suggesting that repair is not intimately responsiblefor their formation. No association was discernable betweenchromosomal aberrations and any of the three methylated purinesstudied.     

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.     

Immunocytochemical identification of DNA adducts, O6-methylguanine and 7-methylguanine, in respiratory and other tissues of rat, mouse and Syrian hamster exposed to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

The present paper reports about an immunocytochemical inventoryof the cell types involved in the metabolic activation of thetobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) to a DNA methylating metabolite. The formation and distributionof the methylated DNA bases O⁶-methylguanine (O⁶-meGua) and7-methylguanine (7-MeGua) were studied in respiratory tissues,oesophagus, liver, kidneys, pancreas, small intestine, colonand prostate of rat, mouse and hamster 6 h after treatment witha single dose of 30 mg NNK/kg. The tissue-and cell-specificdistribution of O⁶-meGua- and 7-meGua-specific nuclear stainingshowed the same patterns and were remarkably similar in rat,mouse and hamster in spite of the diverging spectra of NNK-inducedtumours in these species. In nasal tissue, a target for NNK-inducedtumourigenesis in rat and hamster, but not in mouse, adduct-specificnuclear staining was observed in all three species in sustentacularcells, Bowman glands, respiratory epithelial cells and serousglands. Both methylated DNA bases were also observed in basalcells of the olfactory epithelium of rat and (occasionally)hamster, but not in those of the mouse. In the trachea, a targetfor NNK-induced tumourigenesis in hamster only, substantialadduct-specific nuclear staining was found in basal epithelialand glandular cells of the hamster; in the same cells of ratand mouse only a weak nuclear staining was found. In the lung,a common target for NNK-induced tumourigenesis, the formationof O⁶-meGua and 7-meGua was restricted predominantly to bronchialand proximal bronchiolar epithelium. Nuclear staining in therat was occasionally found in alveolar cells and was also observedin hepatocytes. In the three species investigated, O⁶-meGua-and 7-MeGua-specific nuclear staining was found in target andnon-target tissues. Apparently, and in analogy with resultsobtained in other studies, the species-specific organotropyfor tumour formation of NNK is not exclusively determined byDNA methylation. Expanding methylation data with literaturedata on factors considered to be involved in tumour formation,namely proliferation, toxicity and DNA repair among others,still did not lead to a satisfactory explanation for the species-specificorganotropy observed. Additional factors (yet to be identified),need to be taken into account in order to explain (and predict)tumourigenic effects induced by monofunctional methylating agents.     

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.     

Inhibition by phenylethyl and phenylhexyl isothiocyanate of metabolism of and DNA methylation by N-nitrosomethylamylamine in rats

We investigated the effect of 2-phenylethyl and 6-phenylhexylisothiocyanate (PEITC and PHITC) on the metabolism of the ratesophageal carcinogen, N-nitrosomethylamylamine (NMAA). PEITCwas administered orally to MRC-Wistar rats as single doses of0.1 or 1.0 mmol/kg, or by other regimens. When esophagi andliver slices from the treated rats were incubated with 23 µMNMAA, the formation of 2-to 5-hydroxy-NMAA was inhibited by45–90% for esophagus and by 14–19% for liver slices.In contrast, when esophagi and liver slices from untreated MRC-Wistarrats were incubated in vitro with NMAA and 10 µM PEITC,the PEITC inhibited hydroxy-NMAA formation similarly (by 79–89%)in the two tissues. Also, PEITC inhibited the formation fromNMAA of the hydroxy-NMAAs, formaldehyde and pentaldehyde byesophageal and liver microsomes to similar extents. In studieson DNA methylation by NMAA, 7- and O⁶-methylguanine (O⁶-MeG)were determined by HPLC with fluorimetric detection. Guaninemethylation in esophageal and liver DNA was generally closeto linear for doses of 5–50 mg NMAA/kg. With 50 mg NMAA/kg,guanine methylation in esophageal and liver DNA peaked after5 h, and 8–11% of the peak O⁶-MeG persisted after 72 h.A single dose of 0.1 or 1.0 mmol PEITC/kg reduced the O⁶-MeGlevels by 44–51% in the esophagus but by only 7–22%in the liver. Administration of the PEITC homolog, PHITC, inhibitedNMAA metabolism by liver slices from the treated rats and themethylation of guanine in liver DNA, but had little effect inthe esophagus, i.e. PHITC tended to have the opposite tissuespecificity to PEITC. The finding that administration of PEITCspecifically inhibited NMAA metabolism in the rat esophagussupports the view that PEITC may be a useful chemopreventiveagent against esophageal carcinogenesis in humans.     

Resistance of Salmonella typhimurium TA 1535 to O6-guanine methylation and mutagenesis induced by low doses of N-methyl-N'-nitro-N-nitrosoguanidine: an apparent constitutive repair activity

Salmonella tester strains which are reverted by base-pair substitutionmutagens are relatively insensitive to the mutagenic effectsof N-methyl-N-nitroso compounds. One reason for this insensitivityis the ability of these strains to withstand low doses of thesecompounds before they become sensitive to their mutagenic effects.In this report it is shown that mutagenesis induced by treatmentof Salmonella typhimurium TA 1535 with N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) in buffer is biphasic with a low sensitivity range atlow doses where little mutagenesis occurs, followed by a highsensitivity range whose onset begins after an apparent thresholddose has been exceeded. Levels of O⁶-methylguanine (O⁶-MeG)in the DNA extracted from the bacteria follow a similar dose-responsecurve suggesting a dependency of mutagenesis on O⁶-MeG. In contrast,levels of 7-methylguanine (7-MeG) in the DNA increase linearlywith dose. O⁶-MeG was undetectable at the lowest dose of MNNGwhereas 7-MeG was readily detectable. Although such resistanceto O⁶-alkylation has been demonstrated in MNNG-pretreated (adapted)E. coli, it has not been reported in unpretreated cells. Whenisolated DNA was treated with MNNG a linear dose-response inthe generation of O⁶-MeG was observed. The lack of O⁶-MeG inDNA isolated from MNNG treated cells after low doses is attributedto a saturable, constitutive repair activity in the bacteria.An attempt to observe the removal of O⁶-MeG from the bacteriaafter exposure to a short challenge dose of N-nitroso-N-methylurea(NMU) followed by a subsequent incubation in buffer was unsuccessful,probably because all the repair occurred within the time necessaryto treat and lyse the cells.     

Comparative dosimetry of O6-methylguanine in humans and rodents treated with procarbazine

The accumulation of O⁶-methylguanine (O⁶-meG) in the DNA ofblood leukocytes of 21 Hodgkin's lymphoma patients (followedfor up to 12 cycles of treatment) treated in the context ofMOPP combination chemotherapy with 150 mg procarbazine dailyfor 10 days was examined and compared to that observed in ratstreated with different doses of procarbazine as a single agentonce per day for 10 days. In humans, the adduct accumulatedin a doserelated fashion and appeared to approach a steady-stateafter 7–8 days of treatment Adduct levels on day 10 ofthe treatment cycle averaged 0.25 ± 0.09 (mean ±SD) µmol/molG and, for different individuals, covereda 3-fold range. Intra-individual variability between differenttreatment cycles was much more limited than inter-individualvariability, the two parameters accounting for 8.9% and 84.5%respectively of adduct variance at a constant cumulative dose.Comparison of the dose-response relationships for humans andrats indicates that, under conditions of no depletion of O⁶-alkylguanine-DNAalkyltransferase (AGT), O⁶-meG accumulates in blood leukocyteDNA of humans at a rate which is only approximately 2-fold lowerthan in rats, implying that, to the extent to which O⁶-meG contributesto the genotoxic activity of procarbazine, human susceptibilityto it is likely to be comparable to that of the rat This islikely to be true also of the bone marrow (the tissue of interestas a target tissue for leukaemogenesis), since the tissue distributionof O⁶-meG induced by low doses of procarbazine in rats, miceand rabbits indicated that blood leukocyte levels of this adductclosely reflect those in the bone marrow. Based on these results,it is estimated that by the end of a MOPP chemotherapy cycleO⁶-meG reaches levels of the order of 0.2–0 3 fmol/µgDNA (03–0.5 µmol/molG) in human bone marrow (thetarget tissue of leukaemogenesis observed after such treatment).     

Induction of hprt gene mutations in splenic T-lymphocytes from the rat exposed in vivo to DNA methylating agents is correlated with formation of O6-methylguanine in bone marrow and not in the spleen

The suitability of splenic T-lymphocytes as a substitute tissuefor detection of genotoxic effects induced in vivo by chemicalagents that are organ-specifically activated was tested in ratsexposed to single doses at the potent lung-carcinogen 4-(methylnitrosamino)-1-(3-pyri-dyl)-1-butanone(NNK), acetoxymethylmethylnitrosamine (AMMN) or N-methyl-N-nitrosourea(MNU). NNK, AMMN and MNU methylate DNA most likely via the formationof a methanediazohydroxide ion that decomposes to a methyl diazoniumion.For all three agents, an increase in the levels of O⁶-methylguanineand 7-methylguanine in DNA of rat liver and lung was detectedby reverse phase HPLC and electrochemical detection. Treatmentwith NNK did not result in the formation of O⁶-methylguanineand 7-methylguanine in DNA of bone marrow and spleen, correspondingwith the absence of metabolic activation pathways for this compoundin these tissues. For AMMN formation of both O⁶-methylguanineand 7-methylguanine was detectable in DNA of the spleen, whereasin DNA of bone marrow only very low frequencies of 7-methylguaninewere found at a toxic dose. MNU induced O⁶-methylguanine and7-methylguanine in both spleen and bone marrow. Using splenicT-lymphocytes from the rat no increase above control levelsof the hprt mutant frequencies was found for NNK and AMMN forall exposure levels tested, 32 days after chemical exposure.For MNU a dose-dependent increase in hprt mutant frequency wasfound at exposure levels of 0.097 mmol/kg up to 0.582 mmol/kg.DNA sequence analysis was performed on PCR products of hprtcDNA from 39 MNU-induced 6-thioguanine-resistant T-lymphocyteclones. Single base pair substitutions were found in 25 of thesemutants (64%), GC     

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.     

Chinese hamster cells harbouring the Escherichia coli O6-alkylguanine alkyltransferase gene are less susceptible to sister chromatid exchange induction and chromosome damage by methylating agents

Clones of Chinese hamster V79 cells harbouring the Escherichiacoli O⁶-alkylguanine (O⁶-AG) alkylphosphotriester (AP) alkyltransferase(ATase) gene (clone 8) or a subclone of it that codes only forO⁶-AG ATase activity (clone SB) have been exposed to increasingdoses of N-methyl-N-nitrosourea (MNU) or methybnethanesulphonate(MMS) and the frequencies of induced sister chromatid exchanges(SCEs) measured. In control (clone 2) cells, SCE induction wasalmost linearly proportional to dose of MNU or MMS and at thehighest doses used (15 or 80 µg/ml) SCE frequencies were6 or 8 times background levels, respectively. Slightly lowerlevels of MMS-induced SCEs were seen in clone 8 and clone SBcells whilst, in contrast, MNU-induced SCE levels in these twoclones were drastically reduced being less than twice backgroundlevels at 15 µg/ml. After treatment with N-butyl-N-nitrosourea,SCE frequency was similar in all three clones. At higher doses,MNU treatment produced less chromatid aberrations and micronucleiin clone SB than in clone 2 cells. These results suggest thatATase-repairable damage is involved in the induction of SCE,chromosome aberrations and micronuclei in V79 cells.     

Differential effects of procarbazine and methylnitrosourea on the accumulation of O6-methylguanine and the depletion and recovery of O6-alkylguanine-DNA alkyltransferase in rat tissues

The kinetics of accumulation of the premutagenic DNA adductO⁶-methylguanine (O⁶-meG) in the liver, blood leukocytes, lymphnodes and bone marrow of rats was examined and compared aftersingle or multiple doses of procarbazine, a methylating cytostaticdrug employed in the treatment of Hodgkin's lymphoma patients,and methylnitrosourea (MNU), an experimental methylating agentand carcinogen. Maximal O⁶-meG levels occurred 1–2 h afteradministration of single doses of procarbazine (10 mg/kg) orMNU (1 mg/kg), thereafter decreasing with halflives of     

Detection of O6-butyl- and O6-(4-hydroxybutyl)guanine in urothelial and hepatic DNA of rats given the bladder carcinogen N-nitrosobutyl(4-hydroxybutyl)amine

N-Nitrosobutyl(4-hydroxybutyl)amine (BBN) is a selective bladdercarcinogen in rats. Its organ specificity may depend on severalfactors, including metabolic activation, DNA alkylation andrepair within the target organ. Metabolic activation of BBN,which is asymmetrical, may result in butylating and 4-hydroxybutylatingspecies. To test this view, BBN was administered as a singleoral dose of 20 or 120 mg/rat or six doses of 20 mg/rat over2 weeks. The animals given the single 120 mg dose were killed3, 6 and 24 h after treatment Rats given 20 mg or 6x20 mg BBNwere killed 24 h after the last dose. DNA from liver and urothelialcells was hydrolyzed and analyzed for O⁶- butylguanine (O⁶-BuG)and O⁶-hydroxybutyOguanine [O⁶-(4-OH-Bu)G] as their pentafluorobenzyl-trimethylsilylderivatives by high-resolution gas chromatography—negativeion chemical ionization mass spectrometry with selective ionrecording after immunoaffinity extraction. Polyclonal antibodiesraised against O⁶-(4-hydroxybutyl)- guanosine [O⁶-(4-OH-Bu)GR]were coupled to CNBractivated Sepharose 4B. This was mixed witha gel coupled to antibodies raised against O⁶-BuG, already availablein the laboratory, and the mixed gel was used for the one-stepsample clean-up, enrichment and extraction of O⁶-(4-OH-Bu)Gand O⁶-BuG from hydrolyzed DNA. O⁶- BuG In urothelial DNA ofrats given a single dose of 120 mg BBN increased from 0.44 ±0.12 (µmol/mol guanine (mean ±SE) 3 h after treatment,to 17.9 ± 7.23 µmol/mol guanine at 24 h. O⁶-(4-OH-Bu)Gin the same tissue was 7.7 ± 3.19 (µmol/mol guanine3 h after treatment and 12.2 ± 7.01 µmol/mol guanineat 24 h. O⁶-BuG and O6-(4- OH-Bu)G were always lower in theliver than in urothelialcells. Twenty-four hours after a singledose of 20 mg BBN, urothelial O⁶-BuG was 5.41 ± 1.73(µmol/mol guanine anddid not accumulate after six dosesof 20 mg/rat BBN, since it was 2.59 ± 1.23 (µmol/molguanine 24 h after the last dose. O⁶-BuG in liver DNA was detectableafter the single dose of 20 mg, but not after 6x20 mg/rat BBN.O⁶-(4-OHBu) G was not detected in either the bladder or theliver after 20 mg or after the six doses of BBN. The resultsindicate that both butylating and 4-hydroxybutylating speciesare formed in the target organ DNA of rats given the bladdercarcinogen BBN, but that O⁶-BuG seems to be the lesion mostrelevant to the carcinogenic effect     

Induction of esophageal tumors in zinc-deficient rats by single low doses of N-nitrosomethylbenzylamine (NMBA): analysis of cell proliferation, and mutations in H-ras and p53 genes

Dietary zinc deficiency in rats induces hyperplasia in the esophagus and increases N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumor incidence. Previous work showed a direct relationship between epithelial cell proliferation and esophageal tumor incidence in rats given multiple doses of NMBA. We investigated the effects of single low doses of NMBA in zinc-deficient rats since a single dose of 5.0 mg/kg was reported to be non-carcinogenic in rats. Zinc-sufficient and deficient rats received a single i.g. dose of NMBA at 0.5 or 2.0 mg/kg. At week 14, tumor incidence was 50% with 0.8 +/- 1.0 tumors/rat, and 80% with 2.2 +/- 1.9 tumors/rat, in deficient groups, D(0.5) and D(2.0), that received the lower and higher dose, respectively. In addition, two small papillomas were found in one out of eight untreated zinc-deficient rats. None of the NMBA-treated or untreated zinc- sufficient rats had any tumors. Esophageal cell proliferation, as determined by proliferating cell nuclear antigen (PCNA) immunohistochemistry, showed that, irrespective of NMBA treatment, deficient esophagi had significant increases in the number of labeled cells, the total number of cells, and the labeling index, as compared with zinc-sufficient ones. Mutations in Ha-ras and p53 genes in esophageal tumors were detected by single strand conformation polymorphism (SSCP) analysis. DNA sequencing of variant conformers revealed a point mutation (GGA-->GAA, codon 12) in Ha-ras in 4/5 (80%) and 5/8 (63%) tumors, from D(0.5) and D(2.0) rats, respectively. Three out of eight tumors from D(2.0) rats exhibited SSCP mobility shifts within p53 exons 5 and 7: two tumors (2/8, 25%) had missense mutations and the third, a silent mutation. Of the two tumors with p53 mutations, one had a double mutation (transition at codon 164, TCA-->TTA; transversion at codon 241, AGT-->TGT), and the other tumor, a transition at codon 172 (AGA-->GGA), with amino acid changes in all cases. In parallel with PCNA expression, elevated p53 expression was associated with hyperplastic and dysplastic regions, as well as with tumors, in deficient esophagi. In short, these data indicate that dietary zinc deficiency, with its associated sustained increased cell proliferation in the esophagus, can drive an otherwise non-tumorigenic dose of NMBA into a highly tumorigenic one.      

Quantitation of O6-ethyldeoxyguanosine in ENU alkylated DNA by polyclonal and monoclonal antibodies

Rabbit polyclonal and mouse monoclonal antibodies were developedagainst O⁶-ethylguanosine conjugated with keyhole limpet hemocyanin.Radioimmunoassay (RIA) and a modified enzyme-linked immunosorbantassay (ELISA) were established for the determination of antigen-antibodybinding and quantitation of potentially mutagenic O⁶-ethyldeoxy-guanosine(O⁶-EtdGuo) in DNA treated with ethylnitrosourea (ENU) in vitroand in vivo. Optimum as well as reproducible antibody bindingcould be observed with conjugate concentrations at 0.1 ng/wellimmobilized by overnight drying at 37°C. RIA was several-foldmore sensitive than ELISA in detecting inhibition with O⁶-EtdGuorequiring 0.1 pmol for the 50% inhibition of tracer-antibodybinding. In competitive inhibition assays with polyclonal andmonoclonal antibodies, a linear dose resonse relation was obtainedwith DNA hydrolyzates alkylated in vitro with increasing concentrationsof ENU. Significantly lower modification levels, e.g., 16.0fmol O⁶-EtdGuo, at an O⁶-EtdGuo/dGuo molar ratio of 2.6 x 10^–7in a hydrolyzate of 80 µg rat liver DNA ethylated in vivowith 10 µg ENU/g body weight was determined immunologically.The rate of elimination of O⁶-EtdGuo determined in human fetalkidney epithelial cells treated with 0.65 mM ENU showed that50% of initial O⁶-EtdGuo was removed within 1 h followed bya slow phase of repair with 23% remaining at 8 h post-treatment.     

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.     

Overexpression of c-K-ras, c-N-ras and transforming growth factor {beta} co-segregate with tumorigenicity in morphologically transformed C3H 10T1/2 cell lines

Morphologic transformation and tumorigenicity are separate cellularphenotypes in transformed 1OT1/2 cells. We have investigatedthe levels of expression of genes for c-myc, c-H-ras, c-K-ras,c-N-ras, TGFß and Rb in 42 morphologically transformed1OT1/2 cell lines, in an attempt to define the molecular mechanisntsgoverning morphologic transformation and tumorigenicity in the1OT1/2 cell system. The 101 1/2 cell lines investigated generallyoverexpressed mRNAs for c-myc, c-H-ras, and TGFß relativeto the levels expressed by wild- type 10T1 cells (levels ofexpression >1.5-fold that of wild- type 1011/2 cells). Incontrast, only half of these cell lines overexpressed mRNAsfor c-N-ras and/or Rb relative to wild- type 1OT1/2 cells, andonly 25%; overexpressed c-K-ras mRNA. The mean levels of mRNAexpression for each of c-K-ras, c-N-ras and TGFß genesin tumorigenic cell lines were significantly greater than themean levels of expression in non-tumorigenic cell lines, suggestingan association between twnorigenicity and the levels of expressionof these specific genes. In contrast, levels of expression forc-myc, c-H-ras and Rb genes were not correlated with tumorigenicity.Cell lines that coexpressed high levels of c-K-ras, c-N-rasand TGFß genes were likely to be tumorigenic (11/12cell lines were tumorigenic), whereas cell lines that coexpressedlow levels of these genes were unlikely to be tumorigenlc (1/10cell lines were tumorigenic). High expression of TGFßwas sufficient for tumorigenicity in the absence of high levelsof expression of c-K-ras and c-N-ras (5/5 cell lines were tumorigenic).Elevated expression of either c-K-ras or c-N-ras alone was Insufficientfor tumorlgenlclty, however, coordinate overexpresslon of bothc-K-ras and c-ras was associated with tumorigenicity irrespectiveof the expression status for TGFß (13/15 cell lineswere tuinorigenic). These results suggest that overexpresslonof c-ras, c-H-ras and TGFß are conunonly associatedwith, and possibly mechanistically related to, the process ofmorphologic transformation in 1OTI/2 cells. In addition, theseresults suggest that progression from morphologic transformationto tumorigenicity in 1011/2 cell lines is frequently accompaniedby overexpression of c-K-ras and c-N-ras, and by enhancementof the level of overexpresslon of TGFß     

Chemoprevention of N-nitrosomethylbenzylamine-induced esophageal cancer in rats by the naturally occurring thioether, diallyl sulfide

Diallyl sulfide (DAS) is a principal thioether of garlic (Allium sativum) accounting, in part, for the flavor and fragrance of this herb. Previous studies have shown that DAS is a potent inhibitor of experimentally induced colon cancer in mice. Metabolic studies of other garlic-derived substances suggested that DAS could prevent tumorigenicity of other hepatic activated carcinogens. The present study was designed to determine whether DAS could inhibit the DNA-damaging and tumorigenic effects of N-nitrosomethylbenzylamine in rat esophagus. A dose of 200 mg/kg of DAS given p.o. 3 h prior to N-nitrosomethylbenzylamine administration was found to inhibit the carcinogen-induced nuclear toxicity by 64% to 56% at the two doses (3 and 5 mg/kg) of NMBA tested. These results suggested that the compound was potentially anticarcinogenic. In the carcinogenicity experiment it was found that DAS totally inhibited tumor formation in rats treated with a carcinogenic dose of NMBA (100% inhibition of papilloma and squamous cell carcinoma incidence, P less than 0.0001). Additionally DAS was found to substantially reduce hepatic microsomal metabolism of the carcinogen. These data demonstrate that DAS is unique in its anticarcinogenic activity. It strongly suppresses the tumorigenic effects of potent, metabolically activated monoalkylating carcinogens in the gastrointestinal tract.     

Enzymatic elimination of O6-ethylguanine and stability of O4-ethylthymine in the DNA of malignant neural cell lines exposed to N-ethyl-N-nitrosourea in culture

The capacity of cells for enzymatic repair of DNA structurallyaltered by DNA-reactive agents is of particular interest inrelation to carcinogenesis and with regard to the sensitivity/resistanceof cancer cells towards chemotherapeutic drugs. The developingrat brain is characterized both by a pronounced susceptibilityto the tumorigenic effect of N-ethyl-N-nitroso-urea (EtNU),and by its incapacity, relative to other rat tissues, for enzymaticremoval of 0⁶-ethylguanine from DNA. We have investigated whetherthe latter property is preserved or altered in a panel of ninetumorigenic neural cell lines derived from pre-natal BDIX-ratbrain exposed to EtNUin vivo. Using a competitive radioimmunoassay(RIA) in conjunction with a monoclonal antibody (Mab ER-6) specificfor O⁵-ethyl -2'-deoxyguanosine (O⁶-EtdGuo), the kinetics ofO⁶-EtdGuo elimination from cellular DNA were determined aftera 20-min exposure to EtNUin vitro (100 ug/ml; resulting average‘input’ O⁶-EtdGuo/2'-deoxyguanosine molar ratioin DNA, 0.74 x 10^–5). All of the cell lines showed rapidremoval of O6-EtdGuo from DNA. During a 280-min period of cultureat 37°C following exposure to EtNU, 36–88% of the‘input’ amount of O⁶-EtdGuo was eliminated. Severalof the cell lines removed O⁶-EtdGuo at least as efficientlyas fetal BDIX-rat liver (the most O⁶-EtdGuo elimination-proficientrat tissue thus far documented). In agreement with publisheddata on postnatal rat tissues, O⁴-ethyl-2'-deoxythymidine (O⁴-EtdThd) was not eliminated from DNA to any significant degree ina cell line chosen on the basis of its high O⁶-EtdGuo removalcapacity, as assayed by RIA using a Mab (Mab ER-01) specificfor O-EtdThd. Seven subclones derived from one of the cell linesexhibited considerable variation of O⁶-EtdGuo removal capacity(elimination of 43–93% of the ‘input’ 0⁶-EtdGuowithin 280 min). The relevance of the observed O⁶-alkylguaninerepair-proficiency of malignant neural rat cell lines in relationto carcinogenesis by EtNU in rat brain is not yet clear; however,the apparent instability and intracell population heterogeneityof O⁶-alkylguanine repair capacity in malignant cells deserveattention with respect to chemotherapy with, e.g. chloroethylnitrosoureas.     

Formation and persistence of DNA adducts in organs of CD-1 mice treated with a tumorigenic dose of fluoranthene

Fluoranthene (FA) is tumorigenic to the lung when injected i.p.into CD-1 mice 1, 8 and 15 days after birth (Wang, J.-S. andBusby, W.F.Jr, Carcinogenesis, 14, 1871–1874, 1993). Levels,tissue distribution and persistence of FA-DNA adducts detectedby HPLC-³²P-postlabeling were investigated during the courseof lung tumorigenesis by FA.Anti-10b-N²-deoxyguanosin-1,2,3-trihydroxy-1,2,3,10b-tetrahydrofluoranthene(anti-FADE adduct) was consistently the major adduct in DNAsamples from lung, heart, liver and kidney of animals examinedat different time points from 2 h to 165 days after the lasttreatment with the tumorigenic dose (3.5 mg/mouse) of FA. Severalunidentified adducts were also detected. Lung, the target organfor FA tumorigenicity, contained higher levels of anti-FADEadduct than other tissues from 1–165 days after treatment.The anti-FADE adduct level decreased in a biphasic manner afterreaching maximum values at 2 h in heart and spleen plus thymusand 3 days in lungs, liver and kidneys. About 10% of the maximumamount of anti-FADE adduct remained in lung, liver and heart165 days after final FA treatment, at which time 44% of animalshad developed lung adenomas. Significant inter-litter variations,but no sex differences in adduct levels were observed. Theseresults indicated a positive correlation between anti-FADE adductlevel and persistence in relation to target organ specificityfor tumor formation.     

In vitro and in vivo inhibitory effect of ethanol and acetaldehyde on O6-methylguanine transferase

Human and rat O⁶-methylguanine transferase (O⁶MeGT) are inhibitedin vitro by ethanol at concentrations of 10 to 50 mM and byacetaldehyde, the first metabolite of ethanol, at concentrationsas low as 0.01 µM. Several other enzymes, including glyceraldehyde-3-phosphatedehydrogenase and yeast alcohol dehydrogenase, which like O⁶MeGThave cysteines in their active sites, were not inhibited byacetaldehyde at the levels that inhibited O⁶MeGT. Disulfiram,an acetaldehyde dehydrogenase inhibitor, enhanced the inhibitoryeffect of ethanol in vivo. These results indicate that the inhibitoryeffect of ethanol on O⁶MeGT activity is mediated primarily viaits metabolite, acetaldehyde.