Differential responses of nascent DNA synthesis and chain elongation in V79 and V79/79 cells exposed to u.v. light and chemical mutagens

DNA repair after u.v., N-methyl-N-nitrosourea (MNU) and ethylmethanesulphonate (EMS) in Chinese hamster V79 cells and the mutagensensitive derivative V79/79 was investigated by measurementof five parameters: production of strand breaks in templateDNA, incorporation of [³H]TdR, semi-conservative and repairsynthesis, molecular weights of pulse labelled DNA after mutagenexposure (nascent synthesis) and molecular weights of DNA pulselabelled and chased after mutagen exposure (elongation and ligation).Equal template strand breakage was evident in both cell linesimmediately after MNU and EMS exposure and by 4–5 h afterMNU the extent of fragmentation was greater in V79/79 cells.After u.v. irradiation template fragmentation was evident inV79/79 but not in V79 cells, even though V79/79 cells failedto excise cyclobutane dimers and repair synthesis was demonstablein V79 cells but not in V79/79 cells after exposure to all threemutagens. The rate of incorporation of [³H]TdR during semi-conservativeDNA synthesis was inhibited equally in a dose dependent mannerafter u.v. and MNU exposure; incorporation by V79/79 cells wasinhibited to a greater extent than by V79 cells after EMS exposure.Nascent DNA synthesis was suppressed more in V79/79 cells thanin V79 cells after u.v. but to similar extents in both celllines after MNU and EMS treatment. Pulse chase experiments indicateda lower rate of elongation of nascent DNA in V79/79 cells afterMNU and u.v. exposure but little difference was detectable afterEMS.     

Persistence of DNA single-strand breaks and other tests as indicators of the liver carcinogenicity of 1-nitroso-5,6-dihydrouracil and the noncarcinogenicity of 1-nitroso-5,6-dihydrothymine

The cyclic nitrosourea 1-nitroso-5,6-dihydrothymine [(NDHT) 1-nitrosodihydrothymine] was not significantly carcinogenic when it was administered for 1 year in drinking water (206 mg/liter) to MRC-Wistar rats. In acute toxicity tests, ip injection of saline solutions of 1-nitroso-5,6-dihydrouracil [(NDHU) CAS: 16813-36-8; 1-nitrosohydrouracil], a strong liver carcinogen in rats, produced only mild liver toxicity but marked focal degeneration of myocardial fibers. NDHU injected ip in water solution produced subcapsular liver damage. NDHU, but not NDHT, induced unscheduled DNA synthesis in hepatocyte primary cultures. NDHU, NDHT, and methylnitrosourea [(MNU) CAS: 684-93-5; N-methyl-N-nitrosourea], a liver carcinogen only under special conditions, were tested for their ability, when injected ip into rats, to produce liver DNA damage measured as strand breaks by alkaline sucrose gradient centrifugation. The three nitrosoureas produced similar maximum DNA damage of 2.2-3.2 strand breaks/10(8) daltons. Eighty percent of the damage due to NDHU persisted for 7 days, and the damage at that time was significantly greater than that produced by NDHT and MNU. The varying persistence of liver DNA damage may explain why NDHU, but not NDHT, is a liver carcinogen.     

DNA in proximity to the site of replication is preferentially alkylated in S phase 10T1/2 cells treated with N-methyl-N-nitrosourea

Replicating DNA is more susceptible to modification by N-methyl-N-nitrosourea(MNU), a spontaneously active methylating agent, than bulk DNA.This conclusion is supported by results from two different experimentalapproaches. First, synchronized C3H 10T1/2 clone 8 cells weretreated in S phase with MNU and DNA replicated during the periodof treatment was separated from bulk DNA. This was done by digestingthe purified DNA with restriction enzymes and retaining thereplication fork-associated DNA in nitrocellulose filters. Second,synchronized C3H 10T1/2 clone 8 cells were exposed to 5-bromodeoxyuridineand [³H]MNU and the density-labelled, replicated DNA was separatedin CsCl gradients. Both methods show 2.6 to 5.0 times more [³H]methyladducts per nucleotide residue associated with replicating DNAthan that expected from random methylation. These experimentswere done at low MNU concentrations (0.018 – 0.115 mM)that did not cause any detectable inhibition of DNA synthesisor stimulation of repair replication in 10T1/2 cells.     

Modulation by glutathione of DNA strand breaks induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its aldehyde metabolites in rat hepatocytes.

Activation of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) produced methylating species and two aldehydes: formaldehyde and 4-oxo-4-(3-pyridyl)-butanal (OPB). We investigated the modulation by glutathione of single-strand breaks (SSB) generated by N-methyl-N-nitrosourea (MNU) and the two aldehydes. Hepatocytes were simultaneously exposed to 0.2 mM MNU and to 0-2.00 mM formaldehyde or OPB for 4 h. Both aldehydes induced SSB in a dose-dependent manner. Formaldehyde and OPB exerted a synergistic effect on the formation of DNA SSB by MNU. It is postulated that both aldehydes interfere with DNA repair processes and thus increase the genotoxic effect of DNA methylating species. We investigated whether glutathione (GSH) could protect DNA from NNK-derived intermediates. Formaldehyde (2 mM) and OPB (2 mM) decreased intracellular GSH contents to 60 and 86% of control respectively. DL-Buthionine-[S,R]-sulfoximine (BSO) treatment reduced the GSH contents of hepatocytes to 19% of control but did not reduce the content of cytochrome P450 nor the metabolism of NNK. The frequency of DNA SSB induced by NNK, formaldehyde or OPB was significantly higher in GSH-depleted hepatocytes. GSH repletion with GSH monoethyl ester returned NNK-induced SSB to its initial frequency. OPB but not NNK nor formaldehyde induced double-strand breaks. We conclude that OPB and formaldehyde inhibit the repair of DNA damage induced by methylating species and that GSH reduces the level of DNA damage induced by NNK-derived reactive metabolites.     

Postreplication repair of alkylation damage to DNA of mammalian cells in culture.

Incorporation and alkaline sucrose sedimentation studies of DNA from mouse L-cells have demonstrated the following effects of N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS). Increasing the concentration of both agents increases the number of single-strand breaks or alkali-labile lesions of existing DNA, which affects the incorporation of [3H]thymidine into DNA by reducing its relative rate. DNA that is newly synthesized during the 1st hr in [3H]thymidine after MNU treatment is of lower molecular weight than is existing DNA with alkali-labile lesions in treated cells and is also lower than DNA synthesized in control cells. Such small segments formed in treated cells are elongated and joined to form high-molecular-weight DNA in the subsequent 4-hr chase in thymidine or 5-bromo-2'-deoxyuridine. Near-ultraviolet photolysis selectively degrades 5-bromo-2'-deoxyuridine-elongated DNA to segments that are nearly as small as those before chase. Further, caffeine (2 mM) present during the thymidine chase prevents nascent-strand elongation, although caffeine-insensitive chain growth occurs partly in MNU-alkylated cells. The MMS lesion (single-strand breakage in alkali) in existing DNA also temporarily interrupts replicative synthesis and makes short segments, but their elongation seems insensitive to caffeine. Our results indicate that MNU may produce both caffeine-sensitive interruptions (probably gaps), as ultraviolet damage does, and apurinic site-directed, caffeine-insensitive interruptions in nascent strands, while MMS may cause exclusively the latter. Further evidence for this is the caffeine potentiation of only MNU killing, like ultraviolet killing, of L-cells. The extent of such a specific MNU lesion is estimated to be no more than 4% of the total extent of methylation, predicting that the lesion that is accessible to caffeine-sensitive repair will be a minor product(s) other than N7-methylguanine. Mutagenic and carcinogenic effects of MNU, which are higher than those of MMS, could be ascribed to such a particular MNU lesion(s) and its repair.     

DNA adduct formation and unscheduled DNA synthesis in rat esophagus in vivo after treatment with N-methyl-N-nitrosourea

An in vivo method for assessment of DNA adduct formation and unscheduled DNA synthesis (UDS) in the esophagus of rats was devised. Small ventral incisions were made in the neck and upper abdomen regions of 6 week old F344 rats and ligation of the esophagus with thread at the two extreme ends performed to make an esophageal pouch. For the DNA adduct formation study, a solution (0.5 ml) containing various concentrations of N-[3H]methyl-N-nitrosourea ([3H]MNU) was injected into the pouch. DNA binding levels were calculated from radioactivity of the isolated DNA and dose-dependent DNA adduct formation could be detected 2 h after the treatment with MNU. By HPLC analysis, both 7-methylguanine (7-mGua) and O6-methylguanine (O6-mGua) adducts were identified in the esophageal DNA, the ratio of 7-mGua/O6-mGua being 5.7-12:1. For UDS measurement, a solution containing MNU plus [3H]thymidine (200 microCi/ml) was similarly injected into the pouch. UDS was dose-dependently demonstrated as silver grains over the nuclei of the epithelial cells by autoradiography. The results thus showed that MNU, when injected into the esophageal lumen, can penetrate the surface mucosa, react with the epithelial cell DNA and induce DNA adduct formation and DNA repair synthesis dose-dependently.     

Effects of adriamycin on supercoiled DNA and calf thymus nucleosomes studied with fluorescent probes

The interaction of the antitumor drug Adriamycin with nucleotides, polynucleotides, RNA, calf thymus nucleosomes, and DNA (including pBR322 supercoiled DNA) has been studied using fluorescent probes. The lanthanide terbium is known to interact with guanine and xanthosine to produce high fluorescence enhancement. The nature of the interaction of the lanthanide with the heterocyclic ring in guanine appears to involve both the C-2 and N-7 groups. A striking decrease in fluorescence enhancement was observed with all of the polynucleotides, RNA, DNA, and nucleosomes after treatment with Adriamycin at molar ratios of 1:200 or less. It appears that Adriamycin interacts with the guanine ring, displacing or preventing terbium access to its second site of binding. However, with supercoiled DNA and nucleosomes, the displacement followed a destabilization of the helix at very low drug concentrations. The binding affinities of calf thymus DNA, pBR322 DNA, and calf thymus nucleosomes at 37 degrees for Adriamycin were of the same order of magnitude. Reaction with N-pyrene maleimide, a fluorescent probe which binds to histone H3, showed that Adriamycin interacted with the nucleosome to increase the binding of the probe (only, however, at drug ratios far greater than those required to produce effects with DNA). No compositional changes of supercoiled or nucleosomal DNA or nucleosomal histones were observed by agarose gel or sodium dodecyl sulfate:polyacrylamide gel electrophoresis, respectively. The classic intercalating agent, ethidium bromide, produced minimal displacement of the lanthanide from DNA, although an effect with RNA at high drug concentrations was observed.     

Detection of in vivo DNA repair synthesis in mouse liver and lung induced by treatment with benzo(a)pyrene or 4-nitroquinoline 1-oxide

We examined in vivo DNA repair synthesis in liver and lung of A/HeJ mice treated with benzo(a)pyrene (BP) or 4-nitroquinoline 1-oxide. To differentiate between the removal of carcinogen metabolite:DNA adducts due to cell turnover and DNA repair, we measured unscheduled DNA synthesis (UDS) in the nonreplicating DNA fraction. Mice were exposed to bromodeoxyuridine pellets 1 hr prior to carcinogen treatment. Immediately following carcinogen exposure, mice received 4 hourly i.v. doses of [3H]thymidine. Mice were sacrificed 5 hr post-carcinogen treatment, and DNA was isolated. Purified DNA was then separated into newly replicated and nonreplicated DNA by ultracentrifugation in alkaline CsCl gradients. BP induced UDS in the liver at p.o. doses of 0.3 and 3.0 mg/mouse, whereas we failed to detect UDS in the lung. However, 4-nitroquinoline 1-oxide, another lung carcinogen, induced a definite repair response in the lung but not in the liver. It is not clear why mouse lung cells have the capacity to repair 4-nitroquinoline 1-oxide-induced damage to DNA and not the damage induced by BP, since both of these lung carcinogens form bulky adducts with DNA. These results demonstrate that (a) the in vivo disappearance of BP metabolite:DNA adducts from the lung of the A/HeJ mouse is due to cell turnover, whereas the disappearance of adducts from the liver is due, in part, to DNA repair and (b) induction of in vivo UDS after treatment with two different lung carcinogens is both tissue and carcinogen dependent in this mouse strain.     

Metabolism and nucleic acid binding of N-hydroxy-4-acetylaminobiphenyl and N-acetoxy-4-acetylaminobiphenyl by cultured human uroepithelial cells.

Metabolic activation of N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP), the proximate carcinogenic metabolites of the human bladder carcinogen 4-aminobiphenyl (ABP), was examined in human uroepithelial cells (HUC). Bioconversion was studied by incubating HUC cultures with [3H]N-OAc-AABP or [3H]N-OH-AABP. Three organo-soluble metabolites, N-OH-AABP, 4-acetylaminobiphenyl (AABP), and ABP were identified in ethyl acetate extracts from cultures exposed to N-OAc-AABP. Similarly, AABP and ABP were characterized as the major metabolites from cultures treated with N-OH-AABP. Incubation of N-OAc-AABP with HUC microsomes in vitro yielded primarily the O-deacetylation product N-OH-AABP. The HUC microsomes also catalyzed the N-deacetylation of N-OAc-[14C]AABP, N-OH-[14C]AABP, and [3H]AABP. The O- and N-deacetylase activities for N-OAc-AABP were 55.9 and 38.2 nmol/mg/min, respectively. These O- and N-deacetylase activities were both blocked by paraoxon. Incubation of [3H]N-OAc-AABP or [3H]N-OH-AABP with HUC microsomes and tRNA or DNA showed that 23.0 and 8.0 nmol of N-OAc-AABP and 74.5 and 25.2 pmol of N-OH-AABP were bound per mg protein/mg RNA or DNA, respectively. In comparison, the acetyl CoA-dependent HUC cytosol-mediated bindings of [3H]N-OH-ABP to RNA and DNA were 801 and 447 pmol/mg nucleic acid/mg protein. The HUC microsome-mediated bindings of N-OAc-AABP and N-OH-AABP to nucleic acids were inhibited by paraoxon, whereas the cytosol-mediated binding of N-OH-ABP was insensitive to paraoxon inhibition. Chromatography of the DNA hydrolysate obtained from the in vitro incubation of [3H]N-OAc-AABP or [3H]N-OH-AABP with HUC microsomes showed N-(deoxyguanosine-8-yl)-4-aminobiphenyl as the major adduct, based on comparison with authentic synthetic standard. These results show that human uroepithelia contain microsomal acetyl transferases that are capable of converting the proximate metabolites N-OAc-AABP and N-OH-AABP of the human bladder carcinogen ABP, to reactive electrophiles that bind to DNA. The occurrence of these acetyl transferases in the target organ of the human bladder carcinogen ABP suggests that metabolic activation of some proximate metabolites of ABP could occur directly in HUC and could play a pivotal role in susceptibility to aryl-amine/acetamide induced human bladder cancers.     

Investigation of cell death induced by N-methyl-N-nitrosourea in cell lines of human origin and implication of RNA binding protein alterations

Methylating agents, a widely used class of anticancer drugs, induce DNA methylation adducts, the most biologically significant being O(6)-methylguanine. The efficacy of these drugs depends on the interplay of three DNA repair systems: base excision repair (BER), methyl-directed mismatch repair (MMR) and direct damage reversal by O(6)-methylguanine-DNA methyltransferase (MGMT). An MGMT-inducible, MMR- and BER-proficient HeLa cell line was treated with different concentrations of N-methyl-N-nitrosourea (MNU), a model S(N)1 methylating agent, analogous to widely used methylating cancer chemotherapeutic drugs, under different expression levels of the repair enzyme (MGMT). MNU induced MGMT-dependent apoptotic cell death. In this particular cellular context, the induction of apoptosis was accompanied by modifications of the RNA binding protein poly(A)polymerase and significant down-regulation of the heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. These results implicate alterations of the above mentioned RNA binding proteins in S(N)1 methylating agent-induced cell death and apoptosis, providing a possible perspective regarding their use as biomarkers of tumor resistance/sensitivity to chemotherapy.     

Nitrosourea interaction with chromatin and effect on poly(adenosine diphosphate ribose) polymerase activity.

Poly(adenosine diphosphate ribose) polymerase, a chromatin-bound enzyme, was stimulated 150 to 200% after treatment of HeLa cells with methylnitrosourea (MNU). In contrast, a slight inhibitory effect on enzyme activity was observed after treatment of cells with various concentrations of chloroethylnitrosoureas. To define precisely the differential effects of nitrosoureas on the enzyme activity, their interactions with chromatin substructure were studied. A nonrandom, in vivo alkylation of chromatin DNA by equimolar concentrations of MNU and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was revealed by digestion of nuclei from drug-treated cells with micrococcal nuclease and DNase I. [methyl-14C]MNU interacted preferentially with the more accessible regions of chromatin, the internucleosome linkers, whereas, the [chloroethyl-14C]CCNU alkylated the nucleosomal core DNA to a greater extent. These two drugs also differed in their extent of covalent modification of histone and nonhistone chromosomal protein. The binding of MNU to histones was greater than of CCNU. CCNU mainly affected nonhistone proteins. This difference in the reactivity of methyl and chloroethyl nitrosoureas with chromatin may relate to their differential effect on poly(adenosine diphosphate ribose) polymerase activity, as well as to their carcinogenic and antitumor properties.     

Preferential binding of the carcinogen benzo[a]pyrene to proteins of the nuclear matrix

Rat liver nuclei or hepatocytes were incubated with the procarcinogen benzo[a]pyrene (B[a]P) and its ultimate carcinogen, anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). When nuclei were fractionated by mild micrococcal nuclease digestion into different chromatin regions to determine the distribution of covalent binding to proteins, there was a much higher level of B[a]P bound to proteins of the non-released fraction than to those of released mono- and oligonucleosomes. When non-released material was further fractionated with 2 M NaCl, the highest level of B[a]P binding was found in the proteins of the salt-insoluble fraction. Electrophoretic analysis of [3H]B[a]P-modified nuclear proteins revealed radioactive species migrating in the regions of histones H1 and H3, high mobility group (HMG) proteins 1 and 2, and various high mol. wt non-histone proteins. The non-released fraction contained prominent B[a]P-modified species migrating in the position of the lamins, major components of the nuclear matrix. To confirm B[a]P modification of nuclear matrix proteins, following exposure to B[a]P or BPDE, nuclei were fractionated by a different procedure into an active chromatin fraction, a bulk chromatin fraction, a high-salt-extracted chromatin fraction and a nuclear matrix fraction. Proteins of the nuclear matrix bound consistently more B[a]P metabolites than those of bulk chromatin. This was true following exposure to B[a]P or both low and high concentrations of BPDE, in contrast to previous data on damage to nuclear matrix DNA. Proteins of active chromatin bound more carcinogen than bulk chromatin proteins at low concentrations of BPDE, but less than bulk chromatin at higher concentrations, in parallel with previous data on DNA damage in active chromatin. The potential significance of B[a]P binding to nuclear matrix proteins is discussed.     

Induction of oxidative DNA damage by arsenite and its trivalent and pentavalent methylated metabolites in cultured human cells and isolated DNA

Even though a well-known human carcinogen the underlying mechanisms of arsenic carcinogenicity are still not fully understood. For arsenite, proposed mechanisms are the interference with DNA repair processes and an increase in reactive oxygen species. Even less is known about the genotoxic potentials of its methylated metabolites monomethylarsonous [MMA(III)] and dimethylarsinous [DMA(III)] acid, monomethylarsonic [MMA(V)] and dimethylarsinic [DMA(V)] acid. Within the present study we compared the induction of oxidative DNA damage by arsenite and its methylated metabolites in cultured human cells and in isolated PM2 DNA, by frequencies of DNA strand breaks and of lesions recognized by the bacterial formamidopyrimidine-DNA glycosylase (Fpg). Only DMA(III) (> or =10 micro M) generated DNA strand breaks in isolated PM2 DNA. In HeLa S3 cells, short-term incubations (0.5-3 h) with doses as low as 10 nM arsenite induced high frequencies of Fpg-sensitive sites, whereas the induction of oxidative DNA damage after 18 h incubation was rather low. With respect to the methylated metabolites, both trivalent and pentavalent metabolites showed a pronounced induction of Fpg-sensitive sites in the nanomolar or micromolar concentration range, respectively, which was present after both short-term and long-term incubations. Furthermore MMA(III) and DMA(V) generated DNA strand breaks in a concentration-dependent manner. Taken together our results show that very low physiologically relevant doses of arsenite and the methylated metabolites induce high levels of oxidative DNA damage in cultured human cells. Thus, biomethylation of inorganic arsenic may be involved in inorganic arsenic-induced genotoxicity/carcinogenicity.     

Inhibition of experimental tobacco carcinogen induced head and neck carcinogenesis

Oral cancer models have attempted to demonstrate inhibition of oral carcinogenesis. These models used synthetic carcinogens, lacked a specific mechanism of activity or used non-physiologic doses for carcinogen or inhibitor. To correct these problems the tobacco and environmental carcinogen, dibenzo[a,l]pyrene (DB[a,l]P) (0.25%, 0.010 microM/application) was painted on the tongue and/or vitamin E acid succinate (VE(as)) (0.43 I.U./0.136 (microM/treatment) administered by gavages to Syrian hamsters (14 animals per group) using physiologic low doses, 5X/week. Oral cytology supplied keratinocytes after 1, 10, or 25 weeks of treatment. Cells were analyzed by flow cytometry/laser scanning cytometry. Initiation (1-6 weeks) was suppressed by reducing DNA damage (oxidation lesions: 8-oxo-dG), and repair (comet, fpg, OGG1, NTH1). Reduction in promotion (6-10 weeks) was identified by depressed proliferation (cell cycle, bromodeoxyuridine incorporation (BrdU)) and aneuploidy (propidium iodide stain). p53 and apoptosis expressions were increased (Sub G(1), mitochondrion activation: Apo 2.7, and nucleosomal formation: mebstain (TUNEL)). VE(as) administration reduced dysplasia (10 weeks) and oral cancer formation at 25 (0/7 vs. 5/7 DB[a,l]P) and 30 weeks (3/7 vs. 6/7 DB[a,l]P). Inhibition of oral carcinogenesis by VE(as) involved reversal of several cellular events that contribute towards oral cancer.     

Characterization of naphtho[1,2-a]pyrene and naphtho[1,2-e]pyrene DNA adducts in C3H10T1/2 fibroblasts

Polycyclic aromatic hydrocarbons (PAHs) are a class of carcinogenic chemicals that are ubiquitous in the environment. Fjord-region naphthopyrene isomers are structurally similar to the potent fjord-region PAH carcinogen dibenzo[a,l]pyrene and thus have the potential to be potent carcinogens. Naphtho[1,2-a]pyrene (N[1,2-a]P) exhibited similar bacterial mutagenicity and morphological cell transforming activity when compared to benzo[a]pyrene (B[a]P), whereas the structural isomer, naphtho[1,2-e]pyrene (N[1,2-e]P) was inactive is these bioassays. In this study, we examined the formation of DNA adducts in C3H10T1/2Cl8 (C3H10T1/2) mouse embryo fibroblasts exposed to N[1,2-a]P or N[1,2-e]P and their respective dihydrodiols. The DNA adducts were characterized by co-chromatography with reaction products from anti-N[1,2-a]P diol epoxide (DE) or anti-N[1,2-e]PDE and polydeoxyadenosine (dAdo) or oligodeoxyguanosine (dGuo). C3H10T1/2 fibroblasts exposed to N[1,2-a]P or N[1,2-a]P-9,10-diol produced both anti-N[1,2-a]P-DE-dAdo and -dGuo adducts with total DNA adduction levels of 22.2 to 33.3 pmol DNA adducts/mug DNA. C3H10T1/2 fibroblasts exposed to N[1,2-e]P produced 2 major and 1 minor adducts. C3H10T1/2 fibroblasts exposed to N[1,2-e]P-11,12-diol produced 2 major adducts. All of the identified adducts were anti-N[1,2-e]PDE-dGuo and -dAdo adducts. While the total DNA adduct level in N[1,2-e]P-11,12-diol-treated fibroblasts was extremely high, 105.9 pmol DNA adducts/mug DNA, the level in N[1,2-e]P-treated fibroblasts was 1.47 pmol DNA adducts/microg DNA. We conclude that lack of biological activity of N[1,2-e]P may be related to its inability to form sufficient amounts of N[1,2-e]P-11,12-diol, which would then be metabolized to sufficient amounts of anti-N[1,2-e]PDE needed to transform these fibroblasts.     

Effect of gallium on DNA synthesis by human T-cell lymphoblasts

We have studied the antiproliferative effects of gallium nitrate in cultured CCRF-CEM lymphoblasts. The 50% inhibitory dose for these cells was 120 microM, and after 24 h at a cytostatic concentration (480 microM) S-phase arrest was observed by DNA flow cytometry. Deoxyribonucleoside triphosphate pools were all reduced (dATP, dGTP, and dCTP by 50%, dTTP by 25%), suggesting inhibition of ribonucleotide reductase. Administration of tracer amounts (0.5 microM) of either [3H]uridine or [3H]deoxyuridine confirmed that DNA synthesis had been inhibited to 20% of control rates by gallium. Further, the flow of the ribonucleoside into the dTTP pool and DNA was selectively reduced compared to that of the deoxyribonucleoside. Gallium decreased the specific activity of dTTP labeled from uridine by 50%, whereas the specific activity of dTTP labeled from deoxyuridine was increased 2.5-fold. Thus counts in DNA derived from [3H]uridine were decreased by more than 80%, while counts in DNA derived from [3H]deoxyuridine were virtually unaltered. Uridine incorporation into RNA was not affected. Gallium did not significantly alter the capacity of permeabilized naive cells to incorporate [3H]dTTP into DNA, while 24-h gallium pretreatment (which increased the percentage of S-phase cells) produced a modest increase in [3H]dTTP incorporation, indicating that any effect of gallium on DNA polymerase alpha is minor. Gallium treatment did not induce or inhibit the repair of DNA single strand breaks. These data demonstrate that gallium inhibits replicative DNA synthesis, with the major specific enzyme target probably being ribonucleotide reductase.     

The role of nitric oxide on DNA damage induced by benzene metabolites

Benzene, a tobacco constituent, is a leukemogen in humans and a carcinogen in rodents. Several benzene metabolites generate superoxide anion (O(2)(.-)) and induce nitric oxide synthase in the bone marrow of mice. We hypothesized that the reaction of nitric oxide (*NO) with O(2)(.-) leads to the formation of peroxynitrite as an intermediate during benzene metabolism. This hypothesis was supported by demonstrating that the exposure of mice to benzene produced nitrated metabolites and enhanced the levels of protein-bound 3-nitrotyrosine in the bone marrow of mice in vivo. In the current study, we investigated the influence of nitric oxide, generated from sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, on DNA strand breaks induced by each single or binary benzene metabolite at different doses and compared the levels of the DNA damage induced by each benzene metabolite in the presence of nitric oxide with the levels of DNA strand breaks induced by peroxynitrite at similar doses in vitro. We found that among benzene metabolites only 1,2,4-trihydroxybenzene (BT) can induce significant DNA damage in the absence of nitric oxide. While 1,4-dihydroxybenzene (HQ), 1,4-benzoquinone (BQ) and 1,2-dihydroxybenzene (CAT) require .NO to induce DNA strand breaks, hydroquinone was the most potent DNA-damaging benzene metabolite in the presence of *NO. The order of DNA breaks by benzene metabolites in the presence of *NO is: Peroxynitrite = HQ > BT > BQ > CAT. The *NO and O(2)(.-) scavengers inhibited DNA damage induced by [HQ+*NO]. Benzene, trans,trans-muconaldehyde, and phenol, do not induce DNA strand breaks either in the absence or presence of *NO. However, adding phenol to [HQ+*NO] leads to greater DNA damage than [HQ+*NO] alone. Collectively, these results suggest that nitric oxide is an important factor in DNA damage induced by certain benzene metabolites, probably via the formation of the peroxynitrite intermediate. Phenol, the major benzene metabolite that does not induce DNA damage alone and is inactive in vivo, synergistically enhances DNA damage induced by potent benzene metabolite in the presence of nitric oxide.